System, method, and device for forming an array of emulsions

ABSTRACT

Systems, methods, and devices for forming an array of emulsions. An exemplary device comprises a frame and at least one or a plurality of separate microfluidic modules mounted to the frame and each configured to form an array of emulsions. In some embodiments, each module may be mounted by snap-fit attachment. The device also may include the same sealing member bonded to a top side of each module and hermetically sealing each of the modules. Another exemplary microfluidic device for forming an array of emulsions comprises a stack of layers bonded together. The stack may comprise a port layer forming a plurality of ports. Each port may have a top rim formed by a protrusion that encircles the central axis of the port. The rims may be coplanar with one another to facilitate bonding of a sealing member to each rim.

CROSS-REFERENCES TO PRIORITY APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/894,836, filed Feb. 12, 2018, now U.S. Pat. No. 11,207,685, which inturn is based upon and claims the benefit under 35 U.S.C. § 119(e) ofU.S. Provisional Patent Application Ser. No. 62/458,429, filed Feb. 13,2017. Each of these priority applications is incorporated herein byreference in its entirety for all purposes.

CROSS-REFERENCE TO OTHER MATERIALS

This application incorporates herein by reference in their entirety forall purposes the following: U.S. Patent Application Publication No.2010/0173394 A1, published Jul. 8, 2010; and U.S. Patent ApplicationPublication No. 2014/0378348 A1, published Dec. 25, 2014.

INTRODUCTION

Various powerful approaches for analyzing a fluid sample begin byforming an emulsion with the sample. In the emulsion, the sample is adispersed phase of droplets each encapsulated by an immisciblecontinuous phase. With a biological sample, the droplets are typicallyaqueous droplets surrounded by an immiscible carrier liquid comprisingoil.

A biological sample can be analyzed for the level of a nucleic acidtarget using an emulsion-based strategy. Before the sample is dividedinto droplets, it is combined with reagents to support amplification ofthe target, such as by the polymerase chain reaction (PCR). An emulsionincluding sample droplets is then formed, with the target present inonly a subset of the droplets. The emulsion then is heated or thermallycycled to encourage amplification of the target in each dropletcontaining at least one copy of the target. A signal is detected fromthe droplets to determine which droplets contain amplified target,allowing the level of the target to be calculated from the fraction ofthe droplets that are positive (or that are negative) for the target, inwhat is described as a digital assay.

Microfluidic devices have been developed to form and collect an array ofemulsions in parallel for digital assays. However, increasing the numberof emulsions in the array, to meet the demand for higher samplethroughput, presents numerous engineering challenges. For example,increasing the size of the array without changing its density can makethe device bulky and costly to manufacture. On the other hand, designinga device that forms a higher density array of emulsions under thecontrol of an instrument generally reduces the size of the device's userand instrument interfaces, which can make the device less user-friendly(such as more difficult to load with sample), and the instrument moreexpensive and less reliable.

SUMMARY

The present disclosure provides systems, methods, and devices forforming an array of emulsions. An exemplary device comprises a frame andat least one or a plurality of separate microfluidic modules mounted tothe frame and each configured to form an array of emulsions. In someembodiments, each module may be mounted by snap-fit attachment. Thedevice also may include the same sealing member bonded to a top side ofeach module and hermetically sealing each of the modules. Anotherexemplary microfluidic device for forming an array of emulsionscomprises a stack of layers bonded together. The stack may comprise aport layer forming a plurality of ports. Each port may have a top rimformed by a protrusion that encircles the central axis of the port. Therims may be coplanar with one another to facilitate bonding of a sealingmember to each rim.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic sectional view of an exemplary emulsion-formingassembly of the prior art, taken before emulsion formation and aftersample fluid for a prospective emulsion has been loaded into an inputwell of the device.

FIG. 2 is another schematic sectional view of the emulsion-formingassembly of FIG. 1, taken as FIG. 1 except near the end of emulsionformation, with the resulting emulsion being collected in an output wellof the device.

FIG. 3 is a schematic sectional view of an exemplary emulsion-formingmodule of the present disclosure mounted to a frame and constructed toform an array of emulsions in response to vacuum applied downstream ofeach site of droplet generation, and taken before emulsion formation andafter sample fluid of a prospective emulsion has been loaded into asample reservoir of the module, in accordance with aspects of thepresent disclosure.

FIG. 4 is another schematic sectional view of the emulsion-formingmodule of FIG. 3, taken as in FIG. 3 except near the end of emulsionformation, with the resulting emulsion being collected in an emulsionreservoir of the module, in accordance with aspects of the presentdisclosure.

FIG. 5 is a schematic sectional view of another exemplaryemulsion-forming module of the present disclosure mounted to a frame andtaken near the end of emulsion formation, with the module constructed toform an array of emulsions in response to pressure applied to samplefluid and carrier fluid upstream of the sites of droplet generation, inaccordance with aspects of the present disclosure.

FIG. 6 is a schematic view of an exemplary system for forming an arrayof emulsions and including an instrument and a microfluidic device, withthe instrument being configured to supply carrier fluid to the deviceand to drive and control emulsion formation inside the device, inaccordance with aspects of the present disclosure.

FIG. 7 is an exploded view of an exemplary microfluidic cartridgeincluding a frame and six separate microfluidic modules mounted to theframe by snap-fit attachment, in accordance with aspects of the presentdisclosure.

FIG. 8 is an assembled view of the microfluidic cartridge of FIG. 7 witheach of the six separate microfluidic modules mounted to the frame.

FIG. 9 is a view of the cartridge of FIG. 8, taken as in FIG. 8 afterattachment of a sealing member to a top side of each of the microfluidicmodules, to cover and seal every port, in accordance with aspects of thepresent disclosure.

FIG. 10 is a plan view of the cartridge of FIG. 8.

FIG. 11 is a fragmentary elevation view of the cartridge of 10, takengenerally along line 11-11 of FIG. 10

FIG. 12 is a plan view of the frame of the cartridge of FIG. 7, taken inisolation from the six microfluidic modules.

FIG. 13 is an elevation view of the frame of FIG. 12, taken generallyalong line 13-13 of FIG. 12.

FIG. 14 is another elevation view of the frame of FIG. 12, takengenerally along line 14-14 of FIG. 12.

FIG. 15 is an isometric view of one of the six microfluidic modules ofthe cartridge of FIG. 7 taken in isolation.

FIG. 16 is a plan view of the microfluidic module of FIG. 15.

FIG. 17 is an elevation view of the microfluidic module of FIG. 15,taken generally along line 17-17 of FIG. 16.

FIG. 18 is an exploded view of the microfluidic module of FIG. 15illustrating each of the monolithic layers of the module.

FIG. 19 is another exploded view of the microfluidic module of FIG. 15,with two adhesive bonding layers removed and with the remaining layerscloser together than in FIG. 18 to illustrate how sample wells formed bya well layer receive intake tubes of an overlying channel layer.

FIG. 20 is a plan view of the well layer of FIGS. 18 and 19.

FIG. 21 is a bottom view of the well layer of FIG. 20.

FIG. 22 is a sectional view of the well layer of FIG. 20, takengenerally along line 22-22 with the section plane not exactly vertical.

FIG. 23 is an elevation view of the channel layer of FIGS. 18 and 19.

FIG. 24 a bottom view of the channel layer of FIG. 23, taken generallyalong line 24-24 of FIG. 23.

FIG. 25 a top view of the channel layer of FIG. 23, taken generallyalong line 25-25 of FIG. 23.

FIG. 26 is a fragmentary view of the channel layer of FIG. 25, takengenerally around the region indicated in FIG. 25 and showing channelsand apertures of the module that are dedicated to a single emulsionformation unit.

FIG. 27 is a plan view of a guide/port layer of the microfluidic moduleof FIG. 15.

FIG. 28 is a bottom view of the guide/port layer of FIG. 27.

FIG. 29 is an isometric, fragmentary view of the guide/port layer ofFIG. 27, taken generally around the region indicated in FIG. 27 andshowing a carrier inlet port, a vacuum port, two pairs of sample ventports, and a pair of sample loading ports, with each of the variousports having a rim formed by a ridge at a top end thereof.

FIG. 30 is a sectional view of the microfluidic module of FIG. 15, takengenerally along line 30-30 of FIG. 15.

FIG. 31 is a fragmentary plan view of the microfluidic module of FIG.16, taken generally around the region indicated in FIG. 16, with acapping layer of the module invisible, and with the view showing anaccess region structured as a lateral notch defined by the guide/portlayer over an emulsion reservoir, to avoid obstructing access to thecapping layer over the reservoir and associated channels.

FIG. 32 is a sectional view of the microfluidic module of FIG. 15, takengenerally along line 32-32 of FIG. 15 after attachment of a sealingmember and operative association with an instrument fluidics head thathas pierced and engaged the sealing member at the carrier inlet port andthe vacuum port of the module.

FIG. 33 is a partially exploded, fragmentary, isometric view of anotherexemplary microfluidic cartridge for forming an array of emulsions, withthe cartridge including a frame attached to and supporting a pluralityof microfluidic modules, with the frame forming sample loading ports,carrier inlet ports, and vacuum ports, and with layers of the cartridgedifferentially fragmented, in accordance with aspects of the presentdisclosure.

FIG. 34 is a partially exploded, fragmentary, elevation view of thedevice of FIG. 33, taken generally along line 34-34 of FIG. 33.

FIG. 35 is a fragmentary, assembled view of the device of FIG. 33 withthe layers differentially fragmented as in FIG. 33.

FIG. 36 is a fragmentary, assembled, plan view of the assembled deviceof FIG. 35, with the layers fragmented as in FIG. 35.

FIG. 37 is a fragmentary plan view of the device of FIG. 36, takengenerally around the region indicated in FIG. 36 and illustratingaspects of a vacuum manifold of the device.

FIG. 38 is a fragmentary, isometric, sectional view of the device ofFIG. 36, taken generally along line 38-38 of FIG. 36.

FIG. 39 is a fragmentary, plan view of the device of FIG. 34, takengenerally as in FIG. 37 and illustrating access regions at which thedevice is locally deformed after emulsion formation to seal eachemulsion reservoir before thermal cycling.

FIG. 40 is a bottom view of still another exemplary microfluidic devicefor forming an array of emulsions, with the device including a frameattached to and supporting a single microfluidic module, and with theframe forming sample ports and vacuum ports and also forming an onboardcarrier reservoir, in accordance with aspects of the present disclosure.

FIG. 41 is a fragmentary top view of the device of FIG. 40 with one ofthe onboard carrier reservoirs partially loaded with carrier fluid.

FIG. 42 is a top view of the device of FIG. 40.

DETAILED DESCRIPTION

The present disclosure provides systems, methods, and devices forforming an array of emulsions. An exemplary device comprises a frame andat least one or a plurality of separate microfluidic modules mounted tothe frame and each configured to form an array of emulsions. In someembodiments, each module may be mounted by snap-fit attachment. Thedevice also may include the same sealing member bonded to a top side ofeach module and hermetically sealing each of the modules. Anotherexemplary microfluidic device for forming an array of emulsionscomprises a stack of layers bonded together. The stack may comprise aport layer forming a plurality of ports and providing any combination ofan array of tapered sample loading ports to guide the inlet/outlet endof a pipette to a respective sample reservoir under each loading port,an array of sample vent ports, a carrier-fluid inlet port, acarrier-fluid vent port, and/or a vacuum/pressure port. Each port mayhave a top rim formed by a protrusion that encircles the central axis ofthe port. The rims may be coplanar with one another to facilitatebonding of a sealing member to each rim.

An exemplary cartridge is provided. The cartridge may comprise a frameand a plurality of separate modules mounted to the frame, optionally bysnap-fit attachment. Each module may have an array of sample reservoirsthat are loadable through overlying sample ports from above the module,a corresponding array of emulsion reservoirs, a carrier port, avacuum/pressure port, and a channel network in fluid communication witheach of the sample and emulsion reservoirs and with the carrier port.The channel network may be configured to form droplets of sample fluidreceived from each sample reservoir and to direct the droplets to acorresponding emulsion reservoir when vacuum/pressure is applied to thevacuum/pressure port and carrier fluid is supplied to the carrier port,optionally under pressure. In some embodiments, the modules may besubstantially identical to one another, and/or each module may have atwo-dimensional array of sample reservoirs and corresponding emulsionreservoirs. In some embodiments, the emulsion reservoirs and/or thesample ports within a single module, or of two or more of the modulescollectively, may be arranged according to an ANSI-SLAS microplatestandard, such as spaced from one another by about 4.5 mm or 9 mm. Insome embodiments, the vacuum/pressure port may be a single vacuum port,and the module may include a vacuum manifold providing fluidcommunication between the vacuum port and each of the emulsionreservoirs of the module. The channel network may lie in a first plane,and the vacuum manifold may lie in a second plane that is spaced fromthe first plane. In some embodiments, the cartridge further may includea sealing member, such as a polymer-coated, metal foil, that is attachedto a top side of each of the modules and hermetically seals the carrierport, the vacuum/pressure port, each sample port, each sample reservoir,and/or each emulsion reservoir, to prevent fluid communication betweeneach sample reservoir and the ambient atmosphere outside the modules.

An exemplary microfluidic device is provided. The device may comprise anassembly of layers bonded to one another, such as face-to-face. Thedevice may have an array of sample reservoirs that are loadable throughoverlying sample ports from above the device, a corresponding array ofemulsion reservoirs, a carrier port, a vacuum port, a vacuum manifoldproviding fluid communication between the vacuum port and each of theemulsion reservoirs, and a channel network in fluid communication witheach of the sample and emulsion reservoirs and with the carrier port.The channel network may be configured to form droplets of sample fluidreceived from each sample reservoir and to direct the droplets to acorresponding emulsion reservoir when carrier fluid is supplied to thecarrier port and vacuum is applied to the vacuum port.

Another exemplary microfluidic device is provided. The device maycomprise an assembly of layers bonded to one another, such asface-to-face. The device may have an array of sample reservoirs that areloadable through overlying sample ports from above the device, acorresponding array of emulsion reservoirs, a carrier port, at least onevacuum/pressure port, and a channel network in fluid communication witheach of the sample and emulsion reservoirs and with the carrier port.Each sample port may include a loading guide configured as taperedopening to guide the inlet/outlet end of a pipette tip to an underlyingsample reservoir. The channel network may be configured to form dropletsof sample fluid received from each sample reservoir and to direct thedroplets to a corresponding emulsion reservoir when carrier fluid issupplied to the carrier port, optionally under pressure, andvacuum/pressure is applied to the at least one vacuum/pressure port. Insome embodiments, the opening of the loading may decrease an area of thesample port, measured parallel to a plane defined by the device, by atleast 50%, 75%, 80%, or 90%, among others, and/or may decrease a widthof the port by at least 50% or 70%, among others. In some embodiments,the channel network is located under a tapered region of the sample portand/or is defined by a different layer(s) of the assembly than theopening of the loading guide. In some embodiments, the channel networkhas a ceiling formed by a capping layer of the assembly, and wherein thetapered region is defined by a guide/port layer that overlies thecapping layer. In some embodiments, the guide/port layer forms thecarrier port and the at least one vacuum/pressure port. In someembodiments, the guide/port layer defines openings forming accessregions that extend from a top side to a bottom side of the guide/portlayer and overlapping the emulsion reservoirs, to facilitate accessthereto through other layers of the assembly. Each of the openings maybe defined by a lateral indentation of the guide/port layer or may bebounded circumferentially in a plane parallel to the layers.

Still another exemplary microfluidic device is provided. The device maycomprise an assembly of layers bonded to one another, such asface-to-face. The device may have an array of sample reservoirs that areloadable through overlying sample ports from above the device, acorresponding array of emulsion reservoirs, a carrier port, at least onevacuum/pressure port, and a channel network in fluid communication witheach of the sample and emulsion reservoirs and with the carrier port.The channel network may be configured to form droplets of sample fluidreceived from each sample reservoir and to direct the droplets to acorresponding emulsion reservoir when carrier fluid is supplied to thecarrier port, optionally under pressure, and vacuum/pressure is appliedto the at least one vacuum/pressure port. An upper layer of the assemblymay form at least a top portion of the carrier port, the at least onevacuum/pressure port, and each sample port. The upper layer may includea body and one or more protrusions projecting from the body. The one ormore protrusions may encircle and form a top rim of the carrier port,the at least one vacuum/pressure port, and/or each sample port. In someembodiments, the body may have an upper surface substantially parallelto a plane defined by the assembly. In some embodiments, a sealingmember may be bonded to a top of the one or more protrusions, tohermetically seal each of the ports. In some embodiments, the one ormore protrusions may encircle and form a top rim of a sample vent portfor each sample reservoir.

An exemplary method of forming an array of emulsions is provided. Themethod may be performed with a microfluidic device and/or an instrumentof the present disclosure. In the method, sample fluid may be loadedinto each sample reservoir of the device through a respective, overlyingsample port. For example, the sample fluid may be loaded with a pipette,which may be operated manually be a user. A tip of the pipette may beguided into a sample reservoir by a tapered guide formed by a guide/portlayer of the device. Some or all of the ports of the device then may behermetically sealed with a sealing member. In some embodiments, theports that are sealed with the sealing member may include anycombination of a carrier port, a vacuum/pressure port, a plurality ofsample loading ports, and a plurality of sample vent ports. The step ofsealing prevents fluid communication between each sample reservoir andthe ambient environment outside the device. The layer may be pierced atthe carrier port, the vacuum/pressure port, and each sample vent port. Asource of carrier fluid may be connected to the carrier port, and avacuum/pressure source may be connected to the vacuum/pressure port.Emulsions may be formed and collected in emulsion reservoirs. Eachemulsion reservoir containing a collected emulsion may be hermeticallysealed, in isolation from each of the other emulsion reservoirs, bydeforming at least one layer of the device, to block fluid flow throughone or more channels of the channel network. The device may be thermallycycled. Droplets of each emulsion may be removed from the correspondingemulsion well and a signal detected from the droplets.

The present disclosure provides a disposable cartridge formed byassembling up to six microfluidic modules into a frame. Any suitableaspects of the cartridge may be compliant with microplate standards(such as ANSI-S LAS standards).

In order to integrate functionality that conforms to existing microplatestandards, a very high level of design optimization and miniaturizationmust be achieved. As a result, the interfaces (e.g., ports) for the userand instrument become small. These interfaces can therefore inducedifficulties in user handling. For example, sample fluid is hard to loadthrough tiny sample ports, and the sample ports are challenging to sealafter loading. Also, the small interfaces can cause errors duringinstrument operation, such as failure to apply vacuum or supply oil.

The present disclosure simplifies and improves the interfaces of theprior art, such as those described in U.S. Patent ApplicationPublication No. 2014/0378348 A1, published Dec. 25, 2014. A new layer,which may be referred to as a Bonded Loading Guide (BLG), may be addedto each microfluidic module during manufacture. The BLG may includefunnel-like ports that provide a large target at the top for receivingthe tip of a pipette, and which taper downward to smaller holes thatcommunicate with the underlying sample reservoirs. This designfacilitates pipetting sample fluid into sample reservoirs of the modulebecause the tapered surface of each port acts as a ramp than can guidedownward travel of the pipette tip to the narrow opening at the bottomof the port. The BLG also may provide a vacuum/pressure port forapplying vacuum/pressure and a carrier port for supplying carrier fluid.Either of these ports may flare upward to provide a larger and morereliable interface at which the instrument can connect a vacuum/pressuresource and a carrier source. The BLG further may form a channel portionof a manifold that allows a single vacuum/pressure port to communicatewith a plurality of emulsion reservoirs or sample reservoirs of themodule. Each port defined by the BLG may have a top rim formed by aprotrusion, which may be a ridge. The protrusion may enable moreefficient bonding of a sealing member over each port, optionally bycollapsing and melting slightly when the sealing member is bonded withapplication of heat and pressure. The BLG also may be designed withappropriate openings to retain access to (1) microfluidic channels thatmay be blocked by deformation (e.g., heat staked) to seal each emulsionreservoir after drop generation and before PCR, and (2) each emulsionreservoir after PCR to enable aspiration of droplets for detection.Further aspects of the present disclosure are described in the followingsections: (I) system overview, (II) methods of forming and processingemulsions, and (III) examples.

I. SYSTEM OVERVIEW

This section provides a short discussion of an emulsion-forming assemblyof the prior art, followed by an overview of an exemplary systemcomprising a microfluidic device and an instrument to drive and controlemulsion formation within the device; see FIGS. 1-6.

FIGS. 1 and 2 schematically depict selected features of anemulsion-forming assembly 80 described in U.S. Patent ApplicationPublication No. 2014/0378348 A1, which is incorporated herein byreference. Assembly 80 can be designed to form an array of emulsions,but only a single emulsion formation unit 82 is shown to simplify theillustration. The assembly has a well component 84 underlying and sealedto a channel component 86. The well component provides a sample well 88and an emulsion well 90 for each emulsion formation unit 82.

The channel component defines microfluidic channels, such as a samplechannel 92, a carrier channel (not visible in this view), and a dropletchannel 94 in a top surface thereof. The channels meet one another at achannel junction (not visible in this view) that creates a site ofdroplet generation. A capping layer 96 of the assembly is sealed to thetop surface of channel component 86 to close the channels by forming aceiling of each channel.

Channel component 86 alone or with capping layer 96 defines variousother structures. For example, the channel component also defines anintake tube 98 that projects downward into sample well 88, to an inlet100 formed at the bottom end of the intake tube (and at a position nearthe bottom of the sample well). Tube 98 is in fluid communication withsample channel 92. A sample port 102 (interchangeably termed asample-loading port) is defined by channel component 86 and cappinglayer 96 directly over sample well 88, to permit sample fluid 104 to beloaded into the sample well 88 with a fluid-transfer device, such as apipette 106. The sample port may be sized to permit the bottominlet/outlet end (i.e., a tip) of pipette 106 to pass through the sampleport. A separate vent port may be formed by the channel component foraccess through the capping layer, or the sample port may function as avent during emulsion formation. The channel component also may form acarrier port (not visible here) through which carrier fluid for eachemulsion is supplied to each carrier channel. The channel componentfurther defines a vertical outflow passage 108 that provides fluidcommunication between a droplet channel 94 and emulsion well 90. Avacuum port 110 also is formed by channel component 86 and providesfluid communication between a vacuum source 112 and emulsion well 90after capping layer 96 is pierced, to permit application of vacuum tothe emulsion well.

FIG. 1 shows sample well 88 loaded with sample fluid 104 before emulsionformation. Assembly 80 is not yet connected to vacuum source 112 or acarrier source 114 (see FIG. 2) that supplies carrier fluid 116.

FIG. 2 shows assembly 80 near the end of formation of an emulsion 118.The emulsion includes droplets 120 of sample fluid 104 encapsulated bycarrier fluid 116. The droplets are formed at the channel junction bysegmenting a stream of the sample fluid, travel along droplet channel94, and enter emulsion well 90 via outflow passage 108.

U.S. Patent Application Publication No. 2014/0378348 A1 also disclosesmore detailed, refined embodiments of an assembly for forming an arrayof emulsions. However, the assembly may have various disadvantages thatneed to be addressed. For example, when attempting to arrange thedroplet generators to fit in a particular footprint and/or with aparticular spacing (such as a microplate spacing of 9 mm or 4.5 mm), thesample ports can become quite small and difficult to load manually, andmay not be reliably sealable. Also, the assembly may be awkward tohandle and may not offer any flexibility in the number of emulsionformation units provided to a user. Furthermore, the ports for vacuumand carrier fluid may not offer a reliable, leak-proof connection, andvent ports and sample ports may not be sufficiently separated from oneanother, thereby increasing the chance of cross-contamination.

FIG. 3 shows a schematic, simplified sectional view of an exemplarymicrofluidic device 140 that makes various improvements to themicrofluidic assemblies disclosed in U.S. Patent Application PublicationNo. 2014/0378348 A1, in order to address some or all of the variousdrawbacks listed above. Device 140, which may be described as acartridge, includes at least one microfluidic module 150 mounted to andsupported by a frame 154. As described further below, frame 154 may bestructured to receive and hold two or more copies of module 150 that aresubstantially identical to one another.

Module 150 corresponds to assembly 80 of FIGS. 1 and 2 and may have anysuitable combination of the elements and features described for assembly80. For example, module 150 is configured to form and collect an arrayof emulsions, optionally in parallel. The module may include and/or besubstantially formed by a stack of layers that are bonded together, suchas face-to-face. Bonding may be achieved by any suitable process, suchas a pressure-sensitive adhesive film, solvent bonding, a liquidadhesive, ultraviolet radiation, or a combination thereof, among others.Each layer may be monolithic or formed of two or more layers that arebonded to each other. The layers may be horizontal when the module isbeing loaded and/or forming emulsions. The layers of module 150 mayinclude any combination of a well layer 156, a channel layer 158, acapping layer 160, and a guide/port layer 162. The well layer alone orin combination with one or more other layers may form a well component164 of the module that provides a separate sample well 88 and a separateemulsion well 90 for each emulsion formation unit 166. Each sample wellmay form at least a lower portion of a sample reservoir 168, and eachemulsion well may form at least a lower portion of an emulsion reservoir170. At least a portion of the top wall of each of the reservoirs may beformed by channel layer 158.

The channel layer alone or in combination with one or more other layersmay form a channel component 172 of module 150. Channels, such as asample channel 174, at least one carrier channel 176, and a dropletchannel 178 may be formed in a top surface of channel component 172, andmay be covered and sealed on top by capping layer 160, which may form aceiling of each channel. Channels 174, 176, 178 may meet one another ata channel junction 180 to create a site of droplet generation. Aseparate channel junction 180 and thus site of droplet generation may beformed for each sample reservoir and corresponding emulsion reservoir.More generally, module 150 may define a channel network 182 that is influid communication with each sample reservoir 168 and each emulsionreservoir 170 and that includes each of the sites of droplet generationformed by the channel network. Channel network 182 may lie substantiallyin a horizontal plane and communicates with an intake tube 184 and anemulsion inlet 186 of each emulsion formation unit 166, as describedabove with respect to assembly 80.

Guide/port layer 162, which interchangeably may be described as a guidelayer or a port layer, offers an improved interface with the user and/orwith an instrument that receives microfluidic device 140. Layer 162 maybe monolithic or formed by two or more layers bonded together. Layer 162may form a separate, dedicated sample loading port 188 (interchangeablycalled a sample port) for each sample reservoir, at least one sharedcarrier inlet port 190 (interchangeably called a carrier port or carrierfluid port) to supply carrier fluid, such as oil, to some or all ofemulsion formation units 166/channel junctions 180 of the module, and/orat least one vacuum/pressure port 192. The vacuum/pressure port may beshared among some or all of the emulsion formation units 166/channeljunctions 180 of the module, and may be downstream, as shown here, orupstream thereof (see below). If downstream, the vacuum/pressure portmay be a vacuum port via which a negative pressure (vacuum) is appliedto each emulsion reservoir of the module. If upstream, thevacuum/pressure port may be a pressure port via which positive(pneumatic) pressure is applied to each sample reservoir of the module.Layer 162 also may form a separate, dedicated sample vent port 194(interchangeably called a vent port) (see FIG. 4) for each samplereservoir. Any of these ports may be described as exterior or externalports of the device.

At least one protrusion, such as protrusions 196 (interchangeably termedridges or raised walls), may form a rim of each port (or only a subsetof the ports) and may encircle a central axis of the port. The tops ofthe protrusions may be coplanar with one another. Each protrusion mayprotrude from and may be elevated with respect to a body 198 ofguide/port layer 162, and optionally from a substantially planar topsurface thereof. Port protrusions 196 allow each port to be sealed morerobustly, as described further below. Body 198 may form a lower sectionof each port, and the at least one protrusion may form an upper sectionof the port. Body 198 may be thicker than protrusions 196, such as atleast about 2, 3, 4, or 5 times thicker, when measured orthogonal to aplane defined by the port layer.

Each sample port 188 may include a tapered region 200 that guides thetip of pipette 106 into sample reservoir 168 (see FIGS. 3 and 4). Thetapered region may be formed by guide layer 162 and may taper toward aneck region 202 of minimum diameter between the tapered region andsample reservoir 168. The area of the sample port at its top may be atleast 2, 3, 4, 5, 7, 10, or 15 times the area at neck region 202, togive the user a much larger target area for the tip of the pipette,which minimizes the chance that the tip will miss the sample port andcontaminate another exposed surface of the module. The slope of thetapered region acts as a ramp that guides the pipette tip to the neckregion.

Guide/port layer 162 also may form at least an upper portion of amanifold 204 (see FIG. 3). The manifold allows a single vacuum/pressureport 192 to communicate with each emulsion reservoir (or samplereservoir) of the module, such that vacuum (or positive pneumaticpressure) can be applied in parallel to all of the emulsion reservoirs(or sample reservoirs) of the module via a single port. Thisconfiguration reduces the likelihood that vacuum or pressure will beapplied unequally to different emulsion formation units 166 due toleakage at one or more vacuum/pressure ports that are dedicated toindividual units 166. In some embodiments, a monolithic guide/port layer162 defines a channel structure 206 of manifold 204 that is closed by anunderlying layer, such as capping layer 160, which forms a floor of thechannel structure. Channel structure 206 may lie in a plane that isabove and parallel to microfluidic channel network 182, which formsdroplets.

Guide/port layer 162 further may define a respective opening 208(interchangeably called an access region) over each emulsion reservoir170. The opening may extend completely through the layer, from a topside to a bottom side thereof, to form a window that iscircumferentially bounded in a horizontal plane, or to form a notch at alateral periphery of layer 162. In any event, the presence of opening208 may cause an area of capping layer 160 to be uncovered and exposed.The exposed area may overlap one or more channels of channel network 182that communicate with the emulsion reservoir, allowing each thesechannels to be blocked by local deformation of the capping layer and/orchannel layer after an emulsion has been collected in the emulsionreservoir. Blocking the channels may fluidically isolate the emulsionreservoir from the rest of the channel network and from other reservoirsof the module, allowing the emulsion reservoir to be heated, such asthermally cycled, to promote nucleic acid amplification in droplets ofthe emulsion contained therein, without excessive fluid loss throughevaporation. The exposed area also may overlie an emulsion outlet port(also called an emulsion outlet) defined by channel layer 158. Forexample, the outlet port may be the same as emulsion inlet 186, or maybe laterally offset from the emulsion inlet, and may be in communicationwith emulsion reservoir 170 separately from the emulsion inlet 186 (seebelow). The emulsion may be aspirated from the emulsion reservoir viathe emulsion outlet after collection of the emulsion and optionalheating. Droplets of the aspirated emulsion may be directed through adetection module that detects a signal from the droplets.

FIG. 4 shows a configuration of device 140 during emulsion formation andcollection. After sample fluid 104 has been loaded into each emulsionreservoir 170 of the module, a sealing member 210 (interchangeablycalled a sealing layer, such as a sealing film) may be placed ontomodule 150 such that the sealing member 210 covers the top of anysuitable combination or all of the ports. For example, sealing member210 may cover each sample loading port 188, each sample vent port 194,carrier port 190, and/or vacuum/pressure port 192 (also see FIG. 3).Sealing member 210 may be attached to guide/port layer 162 at the top ofeach protrusion 196. The sealing member may be bonded to protrusions 196by application of heat and/or pressure. The sealing process may slightlymelt sealing member 210 and/or each protrusion 196 at the interfacebetween sealing member 210 and the protrusion, to hermetically sealmodule(s) 150 at each covered port encircled by a protrusion 196. Insome embodiments, the same sealing member 210 may cover and seal portsof two or more modules 150 of device 140, as described further below.Sealing member 210 may include polymer and/or metal. For example, thelayer may be a heat sealable foil having a metal layer bonded to apolymer layer, with the polymer layer in contact with guide/port layer162. A sealing foil and corresponding sealing instrument arecommercially available, such as from Bio-Rad Laboratories, Inc. The useof a foil may be preferred in some cases, because the foil can tear whenpierced, thereby producing a larger opening for fluid flow, andtransfers less material to instruments that interact with the foil(keeping the instruments cleaner). However, an exclusively polymersealing film may be suitable in some cases. Bonding sealing member 210to module(s) 150 may prevent any fluid communication between the ambientatmosphere outside the device and each sample reservoir 168, eachemulsion reservoir 170, channel network 182, and/or channel structure206 of each module. Accordingly, sample fluid 104 may be stored indevice 140 after the sealing member has been applied, and beforeemulsion formation, for any suitable amount of time, such as overnightat room temperature or with refrigeration. The ability to safely storethe sealed device before emulsion formation may be particularlyadvantageous when an instrument is processing other devices 140 and thusis not currently available to drive emulsion formation and subsequentprocessing of the emulsions. Emulsion formation may be performed insidemodule 150 after an opening has been created through sealing member 210at carrier port 190, vacuum/pressure port 192, and each vent port 194;vacuum source 112 has been fluidically connected to vacuum/pressure port192; and carrier source 114 has been fluidically connected to carrierport 190 (see FIG. 4). Each source may be connected via a couplingmember 212 a or 212 b including a piercing member 214 associated with agasket 216. The piercing member creates the opening and the gasketcreates a fluid-tight seal between the source and the port. In otherembodiments, piercing member 214 may not be associated with gasket 216.In other embodiments, an opening may be created through a guide/portlayer, instead of sealing member 210, to open a carrier port and/or avacuum/pressure port (see Example 2). In any event, vacuum source 112applies vacuum to the vacuum/pressure port, which causes sample fluid104 to be aspirated into channel network 182 from each sample reservoir168 via intake tube 184. The aspirated sample fluid is driven alongsample channel 174 as a sample stream to channel junction 180, at whichthe sample stream is segmented into spatially isolated fluid volumes(droplets 120) encapsulated by carrier fluid 116. The vacuum applied atvacuum/pressure port 192 also may drive flow of carrier fluid 116 to thechannel junction, and/or a carrier pump located upstream of the channeljunction may drive flow of carrier fluid into and/or within module 150.Droplets 120 and carrier fluid 116 then travel through droplet channel178 to the corresponding emulsion reservoir 170 at which an emulsion 118including the droplets surrounded by carrier fluid is collected. Eachemulsion then may be thermally cycled in situ after the containingemulsion reservoir has been hermetically sealed, as described above andbelow. At least a portion of the emulsion then may be aspirated from theemulsion reservoir through an opening created over the outlet for theemulsion reservoir, such as an opening created through capping layer 160and sealing member 210 by piercing the capping layer and the film withthe end of a tube, which is operatively connected to a pump.

FIG. 5 shows a schematic representation of another exemplaryemulsion-forming device 230 including one or more microfluidic modules232 mounted to frame 154. Each module 232 is constructed to form anarray of emulsions 118 (only one is visible here) with a correspondingarray of emulsion formation units 166 (only one is visible here). Theemulsions are formed in response to positive pressure applied to eachsample reservoir 168 and to carrier fluid 116 upstream of each channeljunction 180 at which droplets are generated. A sample pressure source234, which may include a pump, applies pressure to each sample reservoir168 (only one is visible here), either at each sample port 188 (whichthus also may be called a pressure port) or at a separate pressure port.In some embodiments, a single pressure port of module 232 (or device230) may be connected to each sample reservoir 168 via a manifold,similar to that described above for module 150. A carrier pressuresource 236, which may include a pump, applies pressure to carrier fluid116, for example, upstream, at, or downstream of carrier source 114.After sample fluid 104 has been loaded into sample reservoir 168, module232 may be sealed with sealing member 210 as described above for module150. A vent port 238 downstream of each emulsion reservoir 170 may beopened by piercing sealing member 210. Each emulsion reservoir may havea dedicated vent port, or a single vent port may serve a plurality ofemulsion reservoirs via a manifold, which may be located above (orbelow) the microfluidic network for forming droplets.

An emulsion formation unit may include any suitable fluidic structures.The unit may have a sample reservoir to receive and hold sample fluid(e.g., a sample fluid that is at least predominantly liquid). The unitalso may have a channel junction downstream of the sample reservoir. Atthe channel junction, sample fluid (such as a stream of the samplefluid) may be divided to form a plurality of fluid volumes (i.e.,droplets) that are isolated from one another by carrier fluid (e.g., acarrier fluid that is at least predominantly liquid and immiscible withthe sample fluid). The unit further may include an emulsion reservoirdownstream of the channel junction and configured to receive and collectan emulsion having the sample fluid as a dispersed phase (droplets) andthe carrier fluid as a continuous phase. The unit also may include aplurality of channels that meet one another at the channel junction. Asample channel of the channels may be located downstream of the samplereservoir and may be configured to receive the sample fluid from thesample reservoir and to direct the sample fluid to the channel junction.At least one carrier channel of the channels may be located downstreamof a source of carrier fluid and/or a carrier port and may be configuredto direct the carrier fluid to the channel junction. A droplet channelof the channels may be upstream of the emulsion reservoir and downstreamof the sample channel and carrier channel(s), and may be configured todirect the fluid volumes and carrier fluid away from the channeljunction and toward the emulsion reservoir.

FIG. 6 shows a schematic representation of an exemplary system 250 forforming an array of emulsions. The system includes microfluidic device140 (see FIGS. 3 and 4) or microfluidic device 230 (see FIG. 5)), and aninstrument 260 configured to receive and hold the device, and to driveand control emulsion formation by the device. Instrument 260 may have atleast one head, such as heads 262, 264, for interfacing with the devicefluidically, and/or modifying the device structurally.

Fluidics head 262 may include at least one carrier coupling member 212 aand at least one vacuum/pressure coupling member 212b. Carrier couplingmember 212 a may be connected to carrier source 114, and vacuum/pressurecoupling member 212 b may be connected to vacuum source 112 or samplepressure source 234. Coupling members 212 a, 212 b may be configured tocreate fluidic connections to at least one carrier port and at least onevacuum/pressure port, respectively, of at least one module 150 or 232 ofdevice 140 or 230. In some embodiments, coupling members 212 a, 212 bmay be used to make successive fluidic connections to each module 150 or232 of device 140 or 230. In some embodiments, a plurality of couplingmembers 212 a and 212 b may be used in parallel with one or two or moremodules 150 or 232.

Fluidics head 262 also may have a plurality of piercing members 214 toopen ports of module(s) 150 or 232 by piercing one or more layers ofdevice 140 or 230. For example, each piercing member 214 may form anopening in sealing member 210, capping layer 160, guide/port layer 162,or a combination thereof, among others (also see FIGS. 3 and 4). Two ofthe piercing members may be arranged coaxially with gaskets 216 ofcoupling members 212 a, 212 b, as described above and below inExample 1. The piercing members may be different lengths, based on thelayers to be pierced. For example, the piercing member of couplingmember 212 a may be longer than the piercing member of coupling member212 b, because coupling element 212 a may pierce sealing member 210 andcapping layer 160, while coupling member 212 b may pierce only sealingmember 210. Other piercing members 214 may pierce sample vent ports 194and a carrier vent port (described further in Example 1). For example,the fluidics head may have a separate piercing member for each vent portof module 150 or 232. In some embodiments, the piercing members for thevent ports may be carried by a different head of instrument 260 thancoupling members 212 a, 212 b.

A deformer head 264 may seal each emulsion reservoir after emulsionformation. The deformer head may have heated protrusions 266 to deform,by heat and pressure, one or more channels connected to each emulsionreservoir, to block fluid flow through each of the channels to/from theemulsion reservoir. The deformer head may be configured to seal all ofthe emulsion reservoirs of module 150 or 232 in parallel or in two ormore subsets serially. In some embodiments, fluidics head 262 may carryprotrusions 266, which may be heated and pressed tightly against themodule after emulsion formation.

Instrument 260 also may include a drive mechanism 268 to propel movementof heads 262, 264 and device 140 or 230 relative to one another. Thedrive mechanism may produce movement of device 140 or 230, while heads262, 264 remain stationary, or vice versa, among others. Relativemovement may be in three dimensions, indicated at 270.

Carrier pressure source 236 may be configured to apply positive pressuresufficient to drive carrier fluid into device 140 or 230. The leveland/or duration of the positive pressure may be different for device 140versus 230. For device 140, carrier pressure source 236 may applypositive pressure sufficient to drive carrier fluid to each site ofdroplet generation. Application of the positive pressure then may bestopped before application of vacuum with vacuum source 112, because thevacuum source can pull additional carrier fluid into the device on itsown. In contrast, for device 230, positive pressure may be applied bycarrier pressure source 236 before application of positive pressure withsample pressure source 234, and then application of positive pressure bysource 236 may continue while sample pressure source 234 is applyingpositive pneumatic pressure to each sample reservoir, to continue todrive carrier fluid into the device.

Further exemplary aspects of microfluidic device 140 are describedelsewhere herein, such as in Section III. Microfluidic devices and/ormodules of the present disclosure may have any suitable combination offeatures disclosed in U.S. Patent Application Publication No.2014/0378348 A1, published Dec. 25, 2014, which is incorporated hereinby reference.

II. METHODS OF FORMING AND PROCESSING EMULSIONS

This section describes exemplary methods of forming and processingemulsions. The method steps presented in this section may be performedin any suitable order and combination, using any of the systems,instruments, devices/cartridges, and/or modules of the presentdisclosure.

Sample fluid may be loaded into a plurality of sample reservoirs of amicrofluidic cartridge/device by a user or robotically. Sample fluid, asused herein, may be any sample-containing fluid, such as an aqueousfluid. The sample fluid also may contain any suitable reagents,including a label to facilitate detecting an analyte in individualdroplets of the sample fluid. A different sample fluid may be loadedinto each sample reservoir.

The sample may be loaded with a pipette, optionally manually. Aninlet/outlet end of the pipette may be placed into each sample reservoirbefore sample fluid is dispensed. The inlet/outlet end may be providedby a removable/disposable tip of the pipette, and a different tip may beused for each sample reservoir. The inlet/out end of the pipette may beguided into the sample reservoir with a loading guide formed by a layerof the microfluidic device.

A sealing member may be attached to the top side of the microfluidiccartridge/device by the user or robotically. Attachment may be performedby application of heat and/or pressure to the sealing member after thesealing member has been placed on the top side, such that the sealingmember covers a plurality of ports of the cartridge/device. The sealingmember may become bonded to a respective top rim of each port of theplurality of ports and may seal each port to prevent fluidingress/egress at the port.

The device/cartridge may be operatively positioned in an instrument,which may perform the following steps automatically.

The sealing member may be pierced at a plurality of ports, to form anopening at each of the ports. Each opening may be at a carrier inletport, a carrier vent port, a vacuum/pressure port, or a sample ventport.

Carrier fluid may be introduced into a carrier fluid manifold of thecartridge/device (or module thereof) via a carrier inlet port. Thecarrier fluid may be driven into the device by application of pressureto the carrier fluid upstream of the carrier inlet port. The carrierfluid may fill a main carrier line and carrier channels that branch fromthe main carrier line. The carrier fluid may reach each channel junctionof a channel network of the device (or module thereof).

Vacuum or positive pneumatic pressure may be applied to avacuum/pressure port of the cartridge/device (or module thereof). Insome embodiments, the vacuum or pressure may be applied to a singlevacuum/pressure port in fluid communication with each of the emulsionformation units of the cartridge/device (or module thereof). If positivepneumatic pressure is applied, pressure also may be applied to thecarrier fluid upstream of the carrier inlet port, to continue toactively drive carrier fluid to each channel junction during emulsionformation. Emulsions may be formed and collected in parallel in an arrayof emulsion reservoirs. Each emulsion reservoir may be sealed andthermally cycled.

III. EXAMPLES

The following examples describe further aspects and embodiments ofsystems, methods, and devices for forming an array of emulsions. Theseexamples are intended for illustration only and should not limit ordefine the entire scope of the present disclosure. Example 1. Cartridgewith Frame and Microfluidic Modules

This section describes further exemplary aspects of microfluidic device140 of Section I, as embodied by a microfluidic cartridge 300(interchangeably called a microfluidic device) having a non-fluidicframe that holds microfluidic modules 150; see FIGS. 7-32. Selectedstructures and features of cartridge 300 that correspond to thosedescribed above for microfluidic device 140 have been assigned the samereference numbers as in device 140.

FIGS. 7-10 show various views of cartridge 300 without (FIGS. 7, 8, and10) and with (FIG. 9) a sealing member 210 that may hermetically sealthe cartridge. The cartridge includes a frame 154 and a plurality ofmicrofluidic modules 150 mounted to the frame. The modules may besubstantially identical to one another, and may be mounted to the frameby the manufacturer or the user. Once mounted, the modules may or maynot be configured to be removable by the user. The modules may be placedinto the frame from above (i.e., the frame may be top-loading).Alternatively, or in addition, the modules may be loaded into the framefrom the bottom (see Examples 2 and 3) or a side of the frame, amongothers. Each module and the frame may have complementary mating featuresthat mate to produce attachment, such as by a snap-fit, and/or may beattached by bonding, among others. The mating features may include aplurality of deformable tabs 302 received in corresponding openings,such as slots 304. Each tab 302 independently may be provided by module150 or frame 154. In the depicted embodiment, each module 150 includestabs 302 protruding from the body of the module at opposite endsthereof, and more particularly, two tabs 302 at one end and one tab 302at the other end for placement into the corresponding number of slots304 of frame 154, to discourage incorrect mating with the modulereversed (i.e., turned 180 degrees) (see FIGS. 13-16).

Any suitable number of modules 150 may be mounted to frame 154 (seeFIGS. 7, 8, and 10). For example, the frame of cartridge 300 forms sixreceiving sites 306 to hold up to six modules 150 (see FIG. 7), but maybe used with only a subset of these sites filled. The modules may createa larger 2D (D=dimensional) array 308 of emulsion formation units 166from a collection of smaller 1D or 2D arrays 310 of units 166 formed bythe modules individually (see FIGS. 8 and 10). The positions of theemulsion formation units in an array may be defined by the locations ofrespective sample loading ports thereof. More particularly, each sampleloading port may define a loading axis along while a pipette tip can beadvanced through the port into an underlying sample reservoir.

Each array may be a rectangular array having rows and columns arrangedorthogonally to one another. The rows may be uniformly spaced and/or thecolumns may be uniformly spaced. In some embodiments, the rows andcolumns may have the same spacing as one another.

Front-to-back rows of array 308 may be identified with numbers (1-12) onframe 154, and left-to-right rows of array 308 with letters (A-H), orvice versa (see FIGS. 8 and 10). In some embodiments, each module 150may have at least one or at least two rows of exactly eight units 166(see FIG. 7). In some embodiments, cartridge 300 may include or exactlyprovide an 8-by-2, 8-by-4, 8-by-6, 8-by-8, 8-by-10, or 8-by-12 array ofunits 166, each capable of forming a separate emulsion from a differentsample fluid. Array 308 and/or array 310 and/or structures therein(e.g., sample reservoirs 168 and/or emulsion reservoirs 170 (see FIGS. 8and 10)) may be arranged according to a microplate standard (e.g.,American National Standards Institute (ANSI)—Society for LaboratoryAutomation and Screening (SLAS)). For example, emulsion formation units166, sample reservoirs 168, and/or emulsion reservoirs 170 may have asubstantially uniform, center-to-center spacing of within 10% or 5% of4.5 mm, 9 mm, or 18 mm, among others, within each row of an individualmodule 150, between rows of the module, or between rows of differentmodules. A standard spacing of reservoirs facilitates transfer of fluidinto and out of the reservoirs with standardized equipment, such asmulti-channel pipettors for use with microplates.

FIGS. 8 and 10 show the exterior ports of cartridge 300, all of whichmay be accessible from the top side of the cartridge. These ports mayinclude, for each module 150, an array of sample loading ports 188 (onesample port per emulsion formation unit 166) and a corresponding arrayof sample vent ports 194 (a dedicated sample vent port associated witheach sample loading port 188 and sample reservoir 168). The module alsomay provide at least one carrier inlet port 190 at which carrier fluidenters the module, and at least one corresponding carrier vent port 312at which air can escape when being forced out by carrier fluid enteringthe module via carrier inlet port 190. At least one vacuum/pressure port192 also may be formed by the module. Negative pressure in the form ofvacuum (i.e., negative gas pressure) may be applied at thevacuum/pressure port. Accordingly, this port will be called a vacuumport 192 for the remainder of this example.

Carrier inlet port 190, carrier vent port 312, and vacuum port 192 mayhave any suitable relative positions. For example, carrier inlet port190 and carrier vent port 312 may be located near opposite ends of eachmodule 150. Also, carrier inlet port 190 and vacuum port 192 may belocated near the same end of the module or near opposite ends relativeto one another.

Cartridge 300 may have a single carrier inlet port 190, a single carriervent port 312, and/or a single vacuum port 192 for each module 150. Ifthe cartridge contains two or more modules 150, each of these differenttypes of port may be arranged in a respective row, which may be parallelto a lateral top edge of the frame. For example, in the depictedembodiment, carrier inlet ports 190 and vacuum ports 192 are arrangedalong one edge portion of the top side of the cartridge, and carriervent ports 312 along the opposite edge portion of the top side. Thevarious ports of the cartridge, such as carrier inlet ports 190, vacuumports 192, sample vent ports 194, sample ports 188, and carrier ventports 312, may have respective top rims 314 that are coplanar with oneanother (see FIG. 11). Each port may have a rim 314 formed by aprotrusion 196, such as a ridge, which may extend continuously aroundthe port in a horizontal plane to encircle the opening and central axisof the port. One or more of the protrusions may encircle only a singleport, and/or the same protrusion may encircle two or more of the ports(see below).

FIG. 9 shows all of the ports of cartridge 300 covered and sealed by thesame sealing member 210. The sealing member may be bonded to eachprotrusion 196 (see FIGS. 8 and 11) by application of heat and/orpressure to create a circumferential, hermetic seal at each port. Rims314, protrusions 196, and/or the top of a module body 316 from which theprotrusions project, may be located at a higher elevation than the topof frame 154, so that the frame does not interfere with application ofsealing member 210 (see FIG. 11). The seal may be breached by the actionof piercing members, as described in Section I, to open ports inpreparation for emulsion formation, to block channels by deformation,and/or to remove fluid from each emulsion reservoir, among others. Insome embodiments, sealing member 210 may cover and seal only a subset ofthe ports of a cartridge (e.g., see Examples 2 and 3).

FIGS. 12-14 show additional features of frame 154, which may be formedat least predominantly of polymer or metal. The frame may berectangular, with an outer frame portion 318 forming four lateral sides,and a plurality of spanning members 320 each attached to oppositelateral sides of the frame portion. Spanning members 320 may help torigidify the frame. Frame 154 may define various apertures, includinglateral slots 304 for attachment of modules 150, as described above, andreceivers 322 to facilitate manipulation by a robotic cartridge handler.

FIGS. 15-19 show assembled and exploded views of a single module 150 inisolation. The module may include a vertical stack of layers bondedtogether. The layers, from bottom to top, may include a well layer 156,a channel layer 158, a capping layer 160, and a guide/port layer 162.Any of these layers may include or be bonded to one another with anintermediate bonding layer, which may be very thin relative to some orall of the other layers. For example, well and channel layers 156, 158may be bonded to one another using a lower bonding layer 330, andcapping layer 160 may be bonded to guide/port layer 162 with an upperbonding layer 332 (see FIG. 18). Well layer 156 alone or in combinationwith lower bonding layer 330 may be described as a well layer or wellcomponent 164. Channel layer 158 alone or in combination with lowerbonding layer 330 and/or capping layer 160 may be described as a channellayer or channel component 172. Openings extending through the layers,from a top side to a bottom side thereof, determine how the variouslayers may fluidically interface with one another, as described in moredetail below, and partially in U.S. Patent Application Publication No.2014/0378348 A1, published Dec. 25, 2014, which is incorporated hereinby reference.

FIGS. 20 and 21 show top and bottom views of well layer 156. The welllayer may be formed of polymer, such as by injection molding, anddefines an array of sample wells 88 and a corresponding array ofemulsion wells 90. As described in Section I, each sample well 88 formsat least a lower portion of a sample reservoir 168, and each emulsionwell 90 forms at least a lower portion of an emulsion reservoir 170. Aplanar body 334 formed integrally with the wells provides a top bondingsurface. The planar body may have any suitable thickness, such as about1-5 mm, among others. Tabs 302 may project from opposite ends of body334 (or from a different layer of the module, such as channel layer 158or guide/port layer 162, among others), and may be formed integrallywith the body.

FIG. 22 shows a sectional view of well layer 156 taken through a pair ofemulsion wells 90. The plane of the section is only approximatelyvertical, such that a top surface 336 of the well layer is visible. Eachemulsion well 90 may have a pair of baffles 338 extending from an upperportion to a lower portion of the well. Baffles 338 may be spacedhorizontally from one another, at least in an upper portion of well 90,to form a chute 340. The chute guides the incoming emulsion downward inthe well, while restricting lateral migration of the emulsion in acircumferential direction around the inside of the well. Without baffles338, a larger proportion of the emulsion wets the upper surface regionof the well and fails to flow downward to join the collected bulk volumeof the emulsion. As a result, the collected bulk volume is smaller, anddroplets that are separate from the bulk volume when thermal cycling isinitiated can introduce error into assay results. For example, thesedroplets may not be thermally cycled properly or may be more susceptibleto damage (e.g., fusion with one another, shrinkage, fragmentation,etc.).

Each baffle 338 may have any suitable structure. The baffle may be aridge that protrudes inward, such as radially inward, from a bodysurface 342 of the well. The ridge may be elongated, and may extendalong any suitable portion, such as majority or all, of the height ofthe well. The ridge may have a pair of elongated walls 344 that meet oneanother to create an edge 346, which may be relatively sharp. Forexample, the angle between the walls of each baffle may be less than 90degrees, such as less than 80, 70, 60, 50, or 45 degrees, among others.The presence a sufficiently sharp edge restricts the ability of theemulsion to leave the chute in a circumferential direction by wettingpast the sharp edge to outer wall 344 that is outside chute 340.

FIGS. 23-26 show various views of channel layer 158. The channel layermay include a planar body 350 and a plurality of intake tubes 184 (alsocalled sippers or straws) projecting from the bottom surface of body 350(and into respective sample reservoirs of well layer 156 (also see FIG.19)). The channel layer may be formed of polymer, such as by injectionmolding. Body 350 may have any suitable thickness, such as 1-5 mm, amongothers. A microfluidic channel network 182 may be formed by openchannels (as grooves) in a top surface 352 of body 350. The channels arecapped by capping layer 160 (see FIGS. 18 and 19), such that eachchannel is bounded circumferentially (see FIG. 25). In other words,channel layer 158 may form the floor and at least a portion of thelateral side walls of each channel of network 182, and capping layer 160may form at least the ceiling of each channel. (The capping layer mayhave substantially featureless top and bottom surfaces.)

Apertures or passages extending through channel layer 158, between topand bottom sides thereof, provide fluid communication between channelnetwork 182 and reservoirs 168, 170 (also see FIG. 20). A respectivesample aperture 354 and a respective passage 356 are vertically alignedwith each sample reservoir 168. The sample aperture is also alignedwith, and/or optionally considered a lower part of, a sample port 188for the sample reservoir (e.g., see FIG. 8 and below). Passage 356 isarranged laterally to sample aperture 354 and forms an inlet of tube 184near the bottom of the sample reservoir (see FIG. 3 and below). Arespective emulsion inlet 186 and a respective emulsion outlet 358 arevertically aligned with, disposed over, and adjoin each emulsionreservoir 170.

Carrier apertures 360, 362 extending through body 350 of channel layer158 may be vertically aligned with, and, optionally, considered a lowerpart of, carrier inlet port 190 and carrier vent port 312, respectively(see FIG. 25) (also see FIGS. 8 and 10). Apertures 360, 362 may belocated at opposite ends of a main carrier line 364 of channel network182 that feeds oil to each emulsion formation unit 166 of the module(see FIGS. 25 and 26).

FIG. 26 shows a portion of channel network 182 for a single emulsionformation unit 166. The sample reservoir 168 and emulsion reservoir 170of unit 166 under the channel network are shown in dashed outline, whichrepresents the top inside perimeter of each reservoir. A sample channel174, at least one carrier channel (two carrier channels 176 a, 176 b areshown here), and a droplet channel 178 meet one another at a channeljunction 180. The channel junction functions as a site of dropletgeneration at which a stream of sample fluid is segmented by carrierfluid, such as by shearing or flow focusing, to form droplets. Samplefluid is received from a sample reservoir 168 under unit 166 via passage356, which is defined in part by tube 184. Carrier fluid is received incarrier channels 176 a, 176 b from at least one main carrier line 364.The carrier channels of each unit 166 branch from the main carrier lineto create, with the main carrier line, a carrier manifold 366 servingeach emulsion formation unit 166 of the module. Droplets formed atchannel junction 180 travel in droplet channel 178 to emulsion inlet186, which communicates with, and optionally adjoins, an emulsionreservoir 170 under the unit 166. The emulsion collected in emulsionreservoir 170 may be removed later through emulsion outlet 358, aftercapping layer 160 over the outlet has been breached.

Additional features of channel network 182 are also shown here. A vacuumchannel 367 provides fluid communication between and is fluidicallyintermediate an overlying vacuum manifold 204 (see below) and underlyingemulsion reservoir 170. More specifically, an enlarged end region 368 ofvacuum channel 367 is vertically aligned with and adjoins an aperture370 defined by capping layer 160 (several apertures 370 are labeled inFIG. 18). Each aperture 370 communicates with the overlying vacuummanifold as described below. Vacuum from a vacuum source can be appliedto each emulsion reservoir via the vacuum manifold, each aperture 370,and each vacuum channel 367. A deformation region 372 of the channelnetwork that can be deformed to seal emulsion reservoir 170 inpreparation for thermal cycling is marked in dashed outline. Thedeformation blocks fluid flow (and evaporation) from emulsion inlet 186via droplet channel 178 and from emulsion outlet 358 via vacuum channel367.

An exemplary positional relationship between emulsion inlet 186,emulsion outlet 358, and baffles 338 is shown here (also see FIG. 22).Emulsion inlet 186 is vertically aligned with chute 340 created by thebaffles and may be close to the top inside perimeter of emulsionreservoir 170. The baffles may be spaced from one another by a fractionof the circumference of the emulsion reservoir, such by less thanone-half or one-third of the circumference. Emulsion outlet 358 may becoaxial with emulsion reservoir 170, to facilitate reaching the bottomof the reservoir with the inlet end of a pipette when emulsion is beingremoved, such as for travel through a detection module.

FIGS. 27-32 show exemplary aspects of guide/port layer 162 and itsrelationship to underlying layers of module 150 and to instrument 260(also see FIG. 6).

Layer 162 may be formed of polymer, such as by injection molding. Thelayer may define at least an upper or outer portion of each sampleloading port 188 and each sample vent port 194 and may define acorresponding through-hole 374 or 376 that extends through the layerbetween top and bottom sides thereof. Each through-hole 374 may overlap(and, optionally, be arranged coaxially with) a corresponding sampleaperture 354 of channel layer 158 (also see FIGS. 24-26). Through-hole376 may not communicate with channel network 182 of the module exceptvia sample reservoir 168. More particularly, a bottom side of guide/portlayer 162 may define a vent channel 377 of each unit 166 that providesfluid communication between the sample loading port and sample vent port(see FIG. 28). After each sample loading port 188 and each sample ventport 194 is sealed at the top with sealing member 210 (also see FIGS. 9and 11), piercing the sealing member at each vent port acts to vent eachof the sample reservoirs for emulsion formation, with reduced chance ofinstrument contamination and/or cross contamination of sample fluidamong units 166 and/or among modules. More particularly, sample loadingport surfaces that may have been contacted with sample fluid remaincovered with an intact (not breached) region of the sealing member,while the sample vent port that is opened by piercing the sealing memberis generally never contacted with sample fluid during loading.

The minimum diameter of each through-hole 374 or 376 may besubstantially smaller than its maximum diameter or width. For example,the minimum diameter of through-hole 374 may be less than one-half ofone-third the maximum diameter of the through-hole due to a taper. Thistaper guides a pipette tip into the underlying sample reservoir asdescribed above in Section I. Also, or alternatively, the minimumdiameter of through-hole 376 may be less than 50%, 33%, 25%, or 20% ofthe maximum diameter of the through-hole, due to a stepwise decrease (asshown here) and/or a taper. This difference reduces the chance that anycontaminant will enter the sample reservoir via the sample vent port andthat any sample fluid will escape via the sample vent port.

Layer 162 also may define at least an upper portion of carrier inletport 190, vacuum port 192, and carrier vent port 312. Accordingly, thelayer may define a respective through-hole 378, 380, 382 for each ofthese ports. The minimum diameter of each through-hole 378, 380, 382 mayor may not be substantially smaller than its maximum diameter, such asless than two-thirds, one-half, or one-third the maximum diameter.

Through-hole 378 may communicate with a vacuum manifold 204 of themodule 150 (see FIG. 28). The vacuum manifold may be formed at least inpart by a channel structure 206 defined by the bottom side of layer 162,which is covered and sealed from below with an underlying layer, such asupper bonding layer 332 and/or capping layer 160 (also see FIG. 18).Channel structure 206 may have a main vacuum line 384 extending fromthrough-hole 380 and a plurality of side branches 386, namely, one foreach unit 166. Each side branch 386 may overlap and communicate with anaperture 370 defined by capping layer 160 (also see FIG. 18) and endregion 368 of one of vacuum channels 367 (also see FIG. 26).Accordingly, application of vacuum to a single vacuum port 192 of module150 results in vacuum being applied to each of the emulsion reservoirsin parallel via vacuum manifold 204 of the module.

Each of through-holes 378, 380, 382 (and thus ports 190, 192, and 312)may or may not communicate with channel network 182 before any of thelayers of module 150 are pierced. In the depicted embodiment, each ofthrough-holes 378, 382 is separated from corresponding carrier apertures360, 362 of channel layer 158 by capping layer 160. The capping layerthus may need to be pierced via carrier inlet port 190 and carrier ventport 312, as described further below, in order to supply carrier fluidto the carrier manifold of the module. This arrangement minimizes thechance of contamination inside the module.

FIG. 29 shows port protrusions 196 formed by layer 162. Each ofprotrusions 196 may encircle a central axis of a port (and/or acorresponding through-hole defined by layer 162 as described above). Theprotrusion may protrude from a body 198 of layer 162 to form a top rim314 at a border of at least one port. The top rim may, for example, beplanar, or may be rounded in cross section, among others. Body 198 mayhave a top surface 390 that is substantially planar and arrangedhorizontally. At least a subset of the protrusions may encircle only oneport. For example, in the depicted embodiments, protrusions 196 atsample loading ports 188, carrier inlet port 190, vacuum port 192, andcarrier vent port 312 are all separate from one another and sample ventports 194. Also, or alternatively, at least a subset of the protrusionsmay encircle at least two ports. For example, a single protrusion 196encircles each of a pair of adjacent sample vent ports 194. Protrusions196 that are dedicated to single ports reduce the risk of crosscontamination and/or leakage between ports.

FIG. 30 shows a sectional view of module 150 taken through a pair ofsample loading ports 188. Layer 162 forms a tapered region 200 thatguides the end of a pipette into corresponding sample reservoir 168.Tapered region 200 may have any suitable taper, forming an angle of lessthan 70, 60, or 50 degrees with a plane of the module or guide/portlayer. A neck region 202 where sample loading port 188 has a minimumdiameter is located under the tapered region and may be defined byguide/port layer 162, capping layer 160, and/or channel layer 158. Therelative positions of tubes 184 and passages 356 are also shown.Channels of channel network 182 formed by the top side of channel layer158 are too small to be visible in this view.

Port/guide layer 162 also may define a plurality of openings 208 thatoverlap each of units 166, and particularly at least a portion of eachemulsion outlet 358 (see FIGS. 27-29 and 31). The openings may benotches defined by the lateral periphery of layer 162, as shown in thedepicted embodiment, or may be windows that are bordered by layer 162 onall lateral sides of each window (see Examples 2 and 3). FIG. 31 shows aportion of module 150 without capping layer 160. Emulsion outlet 358 anddeformation region 372 are not covered or obstructed by layer 162 due tothe presence of opening 208 (also see FIG. 26).

FIG. 32 shows a fluidics head 262 of instrument 260 operatively engagedwith sealing member 210 of module 150, after the sealing member has beenbonded to guide/port layer 162. Carrier coupling member 212 a has agasket 216 that seals the coupling member to carrier inlet port 190, andvacuum coupling member 212 b has a gasket 216 that seals the couplingmember to vacuum port 192. A piercing member 214 of coupling member 212a has pierced both sealing member 210 and capping layer 160. Carrierfluid flows through gasket 216 around piercing member 214 and intocarrier aperture 360 of channel layer 158, as indicated by arrows at392. Similarly, a (shorter) piercing member 214 of coupling member 212 bhas pierced only sealing member 210. Vacuum drives travel of air out ofvacuum manifold 204, as indicated by arrows at 394.

Example 2. Cartridge with Fluidic Frame

This section describes an exemplary microfluidic cartridge 400(interchangeably called a microfluidic device) having a frame 402providing fluidic features for emulsion formation; see FIGS. 33-39.Structures and features of cartridge 400 that correspond to thosedescribed above for microfluidic device 140 of Section I and/orcartridge 300 of Example 1 have been assigned the same reference numbersas in device 140 or cartridge 300.

Cartridge 400 has a plurality of microfluidic modules 404 bonded toframe 402 in a fluid-tight seal. Modules 404 are constructed generallyas described above for modules 150 in Example 1, except that guide/portlayer 162 of each module is omitted. The guide/port layer is replaced bya vacuum manifold layer 406 and a guide/port layer 408 that are sharedamong modules 404. Vacuum manifold layer 406 is bonded to a top side ofcapping layer 160 of each module 404, and guide/port layer 408 is bondedto a top side of manifold layer 406.

Frame 402 has a pair of legs 410 (only one is visible here) and aplatform 412 supported by the legs, optionally formed integrally withthe legs. Platform 412 provides guide/port layer 408 of the cartridgeand defines various ports over each module 404, namely, an array ofsample loading ports 188, a dedicated carrier inlet port 190, and adedicated vacuum port 192. The platform also may define a dedicatedcarrier vent port for each module 404.

Sample loading ports 188 for the entire cartridge may be sealed with asealing member 210, such as a bondable foil, after sample fluid isloaded into sample reservoirs 168 of modules 404, as described above.However, the sealing member may not overlap and/or seal carrier inletports 190 and vacuum ports 192, which may be located at a lowerelevation than the top of the sample loading ports (see FIG. 33).

Each carrier inlet port 190 may have a membrane 414 that seals the port,to reduce the chance of contamination (see FIG. 38). Membrane 414 may beformed integrally with platform 412 and may be pierced after thecartridge is loaded with sample fluid and when ready for emulsionformation.

Each vacuum port 192 is in fluid communication with a respective vacuummanifold 204 formed over each module. The vacuum manifold communicateswith each emulsion formation unit 166 of the module and has thestructure described above for cartridge 300, except that the lateralside walls and the ceiling of the vacuum manifold are not formed by thesame layer. However, in other embodiments, guide/port layer 408(platform 412) is monolithic and forms the lateral side walls and theceiling of the vacuum manifold (e.g., see Example 3).

Guide/port layer 408 of the frame also defines an array of windows 416,namely, one for each emulsion formation unit 166 of the cartridge (seeFIGS. 36, 37, and 39). Each window 416 is functionally similar to one ofopenings 208 described above, except circumferentially bounded, andprovides an access region where the underlying module 404 is not coveredby layers 406 and 408, thereby exposing capping layer 160 over emulsionoutlet 358 and at deformation region 372 (see FIG. 39), as describedabove.

Example 3. Cartridge with Onboard Carrier Reservoir

This section describes an exemplary microfluidic cartridge 500(interchangeably called a microfluidic device) having a frame 502providing fluidic features for emulsion formation, including an onboardreservoir 504 for carrier fluid 116; see FIGS. 40-42. Structures andfeatures of cartridge 500 that correspond to those described above formicrofluidic device 140, cartridge 300, and/or cartridge 400 have beenassigned the same references numbers as in those devices.

Cartridge 500 has a structure shared generally with cartridge 400,including modules 404 bonded in a fluid-tight seal to an underside of aplatform 506 of frame 502. (Only one module 404 is shown in FIG. 40 tosimplify the presentation.) Platform 506 forms a guide/port layer 508 ofthe cartridge, which has the vacuum manifold 204 of manifold layer 406of cartridge 400 formed integrally by platform 506, rather than as aseparate layer. Platform 506 defines, for each module 404, an array ofsample ports 188 (see FIGS. 41 and 42) and a corresponding array ofwindows 510 (similar to windows 416 of cartridge 400), a carrier inletport 190, and a vacuum port 192 that communicates with a vacuum manifold204. However, rather than receiving carrier fluid from an off-cartridgesource, carrier inlet port 190 of each module receives carrier fluid 116from a carrier reservoir 504 provided by the cartridge over the module.Carrier reservoir 504 has a floor and lateral side walls formed by frame502, and may be covered with a sealing member bonded to a rim 512 (seeFIG. 41). The lateral side wall and rim of the reservoir may be providedby a protrusion 514 that projects upward from a body 516 of platform506. Portions of reservoir rim 512 may be shared with rims 518 ofadjacent sample loading ports 188.

Example 4. Selected Embodiments I

This example describes selected embodiments of the present disclosurepresented as a series of indexed paragraphs.

1. A device to form an array of emulsions, comprising: (A) a frame; and(B) a plurality of separate microfluidic modules mounted to the frame,each module including an array of emulsion formation units in fluidcommunication with one another and configured to form an array ofemulsions, each unit including a sample reservoir to receive samplefluid, an emulsion reservoir to collect an emulsion including dropletsof the sample fluid surrounded by carrier fluid, and a channel junctionat which a sample channel, at least one carrier channel, and a dropletchannel meet one another.

2. The device of paragraph 1, wherein the channel junction is configuredto receive the sample fluid from the sample reservoir via the samplechannel, and carrier fluid from a carrier port via the at least onecarrier channel, and to direct the droplets to the emulsion reservoirvia the droplet channel.

3. The device of paragraph 1 or paragraph 2, wherein each moduleincludes at least one carrier port and at least one vacuum/pressureport, and wherein the emulsion formation units of the module areconfigured to form and collect the array of emulsions when carrier fluidis supplied, and optionally driven under pressure, to the at least onecarrier port and vacuum or positive pneumatic pressure is applied to theat least one vacuum/pressure port.

4. The device of paragraph 3, wherein the at least one carrier port is asingle carrier port, wherein the sample, carrier, and droplet channelsof each emulsion formation unit of the module belong to the same channelnetwork, and wherein the channel network includes a carrier manifoldthat directs carrier fluid from the single carrier port to the channeljunction of each emulsion formation unit of the module.

5. The device of paragraph 3 or paragraph 4, wherein the at least onevacuum/pressure port is a single vacuum/pressure port, and wherein themodule includes a vacuum/pressure manifold configured to communicatevacuum or positive pneumatic pressure applied at the vacuum/pressureport to each sample reservoir or each emulsion reservoir of the module.

6. The device of any of paragraphs 3 to 5, wherein the vacuum/pressureport is a vacuum port.

7. The device of any of paragraphs 1 to 6, wherein each module includesan assembly of layers bonded together, and wherein the assembly createseach emulsion formation unit of the module.

8. The device of paragraph 7, wherein the assembly includes an externalcarrier port and an external vacuum/pressure port.

9. The device of paragraph 7 or paragraph 8, wherein the assemblyincludes an array of sample ports arranged in vertical alignment with anarray of sample reservoirs.

10. The device of any of paragraphs 1 to 9, wherein each module includesa body, one or more protrusions projecting from the body, and aplurality of ports, and wherein the one or more protrusions form a toprim of each port of the plurality of ports.

11. The device of paragraph 10, wherein the one or more protrusions forma top rim of each sample loading port of a plurality of sample loadingports, a top rim of each sample vent port of a plurality of sample ventports, and/or a top rim of a carrier inlet port, and/or a top rim of avacuum/pressure port.

12. The device of paragraph 10 or paragraph 11, wherein each protrusionof the one or more protrusions of each module has a top surface, andwherein the top surfaces of the modules are substantially coplanar withone another.

13. The device of any of paragraphs 1 to 12, further comprising a samelayer bonded to each of the modules.

14. The device of paragraph 13, wherein the layer is a sealing layerthat covers and seals a plurality of ports of each module.

15. The device of paragraph 14, wherein the sealing layer is aheat-sealable foil

16. The device of paragraph 14 or paragraph 15, wherein the sealinglayer is bonded directly to one or more protrusions of each module.

17. The device of any of paragraphs 1 to 16, wherein a top portion ofeach module projects above a top of the frame.

18. The device of paragraphs 17, wherein the top portion includes a bodyof the module and one or more protrusions that project upward from thebody.

19. The device of any of paragraphs 1 to 18, wherein a same sealinglayer hermetically seals all of the modules, such that each of theemulsion formation units of each module is fluidically isolated from theambient atmosphere outside the module.

20. The device of any of paragraphs 1 to 19, wherein the frame isconfigured to support and elevate each module.

21. The device of any of paragraphs 1 to 20, wherein each module ismounted to the frame at least in part by a snap-fit attachment.

22. The device of any of paragraphs 1 to 21, wherein each moduleincludes a plurality of tabs received in corresponding apertures of theframe.

23. The device of paragraph 22, wherein each module is elongated betweenopposite ends thereof, and wherein each of the opposite ends includes atleast one of the tabs.

24. The device of any of paragraphs 1 to 23, wherein the device includes96 sample reservoirs and 96 emulsion reservoirs each arranged in an8-by-12 array.

25. The device of any of paragraphs 1 to 24, wherein a spacing of samplereservoirs and/or emulsion reservoirs within each module is about thesame as a spacing of sample reservoirs and/or emulsion reservoirsbetween adjacent modules.

26. The device of paragraph 25, wherein the spacing is within 10% of 9mm.

27. The device of paragraph 1, wherein the frame forms a top rim of aplurality of ports of each module.

28. The device of paragraph 27, wherein the frame forms a pierceablemembrane sealing one or more ports of each module.

29. The device of any of paragraphs 1 to 28, wherein each samplereservoir is vertically aligned with a sample port, and wherein thesample port includes a tapered region configured to guide aninlet/outlet end of a pipette into the sample reservoir.

30. The device of any of paragraphs 1 to 29, wherein the modules aresubstantially identical to one another.

31. The device of any of paragraphs 1 to 30, wherein the frame isconfigured to receive at least three of the modules, and, optionally,only six of the modules.

32. The device of any of paragraphs 1 to 31, wherein each module has atwo-dimensional array of emulsion formation units.

33. The device of any of paragraphs 1 to 32, wherein the device includesa vacuum port and vacuum manifold for each module, and wherein thevacuum port is in fluid communication with each emulsion reservoir ofthe module via the vacuum manifold.

34. The device of paragraph 33, wherein the channels of each module liein a first plane, and wherein the vacuum manifold lies in a second planethat is parallel to and spaced from the first plane.

35. The device of any of paragraphs 1 to 34, wherein the device has arow of carrier inlet ports and a row of vacuum/pressure ports, andwherein the rows are parallel to one another.

36. The device of paragraphs 35, wherein each module includes adifferent one of the carrier inlet ports and a different one of thevacuum/pressure ports.

37. A device to form an array of emulsions, comprising: an assembly of aplurality of layers bonded together, the assembly creating an array ofemulsion formation units in fluid communication with one another andconfigured to form an array of emulsions, each unit including a samplereservoir to receive sample fluid, an emulsion reservoir to collect anemulsion including droplets of the sample fluid surrounded by carrierfluid, and a channel junction at which a sample channel, at least onecarrier channel, and a droplet channel meet one another.

38. The device of paragraph 37, wherein the channel junction isconfigured to receive sample fluid from the sample reservoir via thesample channel, and carrier fluid from a carrier port via the at leastone carrier channel, and to direct droplets to the emulsion reservoirvia the droplet channel.

39. The device of paragraph 37, wherein the assembly includes avacuum/pressure port and a vacuum/pressure manifold.

40. The device of paragraph 39, wherein the assembly includes a carriermanifold lying in a different plane than the vacuum/pressure manifold.

41. The device of any of paragraphs 37 to 40, wherein the assemblyincludes a respective sample loading port located over each samplereservoir, and wherein the sample loading port includes a tapered regionconfigured to guide an inlet/outlet end of a pipette into the samplereservoir.

42. The device of paragraph 41, wherein the sample loading port includesa neck region located under the tapered region, and wherein the neckregion is located between the tapered region and the sample reservoir.

43. The device of paragraph 41 or paragraph 42, wherein the sampleloading port tapers such that a maximum cross-sectional area of thesample loading port is at least 2, 3, 4, or 5 times a minimumcross-sectional area of the sample loading port.

44. The device of any of paragraphs 41 to 43, wherein the sample loadingport has a maximum diameter that is at least 2 or 3 times a minimumdiameter of the sample loading port.

45. The device of any of paragraphs 41 to 44, wherein a cross-sectionalshape of the sample loading port changes between a top and a bottom ofthe tapered region.

46. The device of any of paragraphs 41 to 45, wherein one of the layersof the assembly defines a plane, and wherein at least a portion of thetapered region forms an angle of less than 70, 60, or 50 degrees withthe plane.

47. The device of any of paragraphs 41 to 46, wherein each sampleloading port has a top rim formed by a protrusion.

48. The device of any of paragraphs 37 to 47, wherein each emulsionreservoir includes a pair of downwardly-extending baffles.

49. The device of paragraph 48, wherein a chute is formed between thebaffles.

50. The device of paragraph 48 or 49, wherein emulsion fluid enters theemulsion reservoir via an inlet that is vertically aligned with thechute.

51. The device of any of paragraphs 48 to 50, wherein each baffle has apair of walls that meet one another at an edge, and wherein the pair ofwalls form an angle of less than 90, 80, 70, 60, or 50 degrees.

52. The device of any of paragraphs 48 to 51, wherein each baffleextends from a top region to a bottom region of the emulsion reservoir.

53. The device of any of paragraphs 48 to 52, wherein the baffles arespaced from one another near a top of the emulsion reservoir by lessthan one-third the inside circumference of the emulsion reservoir.

54. The device of any of paragraphs 37 to 53, wherein the assemblyincludes a body, one or more protrusions projecting from the body, and aplurality of ports, and wherein the one or more protrusions form a toprim of each port of the plurality of ports.

55. The device of paragraph 54, wherein the one or more protrusions forma top rim of each sample loading port of a plurality of sample loadingports, and/or a top rim of a carrier inlet port, and/or a top rim of avacuum/pressure port, and/or a top rim of each sample vent port of aplurality of sample vent ports.

56. The device of paragraph 54 or paragraph 55, wherein the one or moreprotrusions are a plurality of protrusions each having a top surface,and wherein the top surfaces are coplanar with one another.

57. The device of any of paragraphs 54 to 56, wherein each protrusionforms part of a port and encircles a central axis of the port.

58. The device of any of paragraphs 57, where the port includes anopening that extends into the assembly from a periphery thereof, andwherein the protrusion bounds a top portion of the opening.

59. The device of any of paragraphs 54 to 58, wherein the one or moreprotrusions include a plurality of protrusions, wherein the assemblyincludes a respective sample loading port for each sample reservoir, andwherein a different protrusion of the plurality of protrusions forms arim of each respective sample loading port.

60. The device of any of paragraphs 54 to 59, further comprising asealing layer bonded directly to the one or more protrusions.

61. The device of paragraph 60, wherein the sealing layer covers andseals a plurality of ports of the assembly.

62. The device of paragraph 61, wherein the sealing layer is a film thatincludes foil.

63. A device to form an array of emulsions, comprising: (A) an array ofsample reservoirs; (B) an array of sample ports overlying and alignedwith the array of sample reservoirs; (C) an array of emulsion reservoirscorresponding to the array of sample reservoirs; (D) a carrier port; (E)a vacuum/pressure port; (F) a vacuum/pressure manifold in fluidcommunication with each emulsion reservoir; and (G) a channel network influid communication with each of the sample and emulsion reservoirs andwith the carrier port and configured to form droplets of sample fluidreceived from each sample reservoir and direct the droplets to acorresponding emulsion reservoir when carrier fluid is supplied to thecarrier port and vacuum or positive pneumatic pressure is applied to thevacuum/pressure port; wherein the device includes a plurality of layersbonded together and forming the sample reservoirs, the sample ports, theemulsion reservoirs, the carrier port, the vacuum port, the vacuummanifold, and the channel network.

64. The device of paragraph 63, wherein the channel network lies in afirst plane, and wherein the vacuum manifold includes a channelstructure lying in a second plane that is vertically offset from thefirst plane.

65. The device of paragraph 64, wherein the sample ports have inletslocated on a top side of the plurality of layers bonded together, andwherein the channel structure is closer to the inlets than the channelnetwork is to the inlets.

66. The device of paragraph 65, wherein a top layer of the plurality oflayers forms the inlets of the sample ports and a ceiling of the channelstructure.

67. The device of paragraph 66, wherein the top layer is a monolithiclayer including a bottom surface having a recess formed therein, andwherein the recess forms side walls of the channel structure.

68. The device of paragraph 66 or paragraph 67, wherein the top layerforms a rim of the vacuum port.

69. The device of paragraph 68, wherein the top layer includes a body,and wherein the rim is formed by a ridge that protrudes from the body.

70. The device of paragraph 69, wherein the body has a substantiallyplanar surface, and wherein the ridge protrudes from the planar surface.

71. The device of any of paragraphs 66 to 70, wherein the top layerdefines a tapered portion of each sample port, and wherein the taperedportion tapers toward an underlying sample reservoir.

72. The device of any of paragraphs 66 to 71, wherein the top layer is amonolithic layer that forms a rim of the carrier port, a rim of eachsample port, and a rim of the vacuum port.

73. The device of paragraph 72, wherein the top layer forms a rim ofeach vent portion of a plurality of vent ports, and wherein each ventport communicates with one of the sample reservoirs via a correspondingsample port.

74. The device of paragraph 73, wherein the rims of the sample ports,carrier port, and vacuum port are coplanar with one another.

75. The device of paragraph 74, wherein the rims of a plurality of ventports are coplanar with the rims of the sample ports, carrier port, andvacuum port.

76. The device of any of paragraphs 66 to 75, wherein the plurality oflayers includes a capping layer that forms a ceiling of the channelnetwork, and wherein the top layer does not cover a portion of thecapping layer over each emulsion reservoir.

77. The device of paragraph 76, wherein the capping layer defines aplurality of notches or through-holes, and wherein each notch orthrough-hole is aligned with the portion of the capping layer over oneof the emulsion reservoirs.

78. A device to form an array of emulsions, comprising: (A) an array ofsample reservoirs; (B) an array of sample ports overlying and alignedwith the array of sample reservoirs; (C) an array of emulsion reservoirscorresponding to the array of sample reservoirs; (D) a carrier port; (E)a vacuum port; and (F) a channel network in fluid communication witheach of the sample and emulsion reservoirs and with the carrier port andconfigured to form droplets of sample fluid received from each samplereservoir and direct the droplets to a corresponding emulsion reservoirwhen carrier fluid is supplied to the carrier port and vacuum is appliedto the vacuum port; wherein the device includes a plurality of layersbonded together and forming the sample reservoirs, the sample ports, theemulsion reservoirs, the carrier port, the vacuum port, and the channelnetwork; and wherein each sample port tapers toward a correspondingsample reservoir.

79. The device of paragraph 78, wherein the sample port includes atapered region and a neck region, and wherein the neck region is locatedbetween the tapered region and the sample reservoir.

80. The device of paragraph 78 or paragraph 79, wherein the sample porttapers such that a maximum cross-sectional area of the sample port is atleast 2, 3, 4, or 5 times a minimum cross-sectional area of the sampleport.

81. The device of any of paragraphs 78 to 80, wherein the sample porttapers such that a maximum diameter of the sample port is at least 2 or3 times a minimum diameter of the sample port.

82. The device of any of paragraphs 78 to 81, wherein a cross-sectionalshape of the sample port changes between a top and a bottom of a taperedregion of the sample port.

83. The device of any of paragraphs 78 to 82, wherein one of the layersdefines a plane, and wherein a wall region of the sample port forms anangle of less than 70, 60, or 50 degrees with the plane.

84. The device of any of paragraphs 78 to 83, wherein each sample portincludes an opening encircled at a top region thereof by a respectivecontinuous ridge.

85. The device of any of paragraphs 78 to 84, wherein a tapered regionof the sample port is formed by a different layer or layers than thechannel network.

86. The device of any of paragraphs 78 to 85, wherein a top, monolithiclayer of the layers forms a rim and a tapered portion of each sampleport, a rim of the carrier port, and a rim of the vacuum port, andwherein, optionally, the layer is injection molded.

87. The device of paragraph 86, wherein the layer forms a rim of arespective vent port for each sample reservoir.

88. A device to form an array of emulsions, comprising: (A) an array ofsample reservoirs; (B) an array of sample ports overlying and alignedwith the array of sample reservoirs; (C) an array of emulsion reservoirscorresponding to the array of sample reservoirs; (D) a carrier port; (E)a vacuum port; and (F) a channel network in fluid communication witheach of the sample and emulsion reservoirs and with the carrier port andconfigured to form droplets of sample fluid from each sample reservoirand direct the droplets to a corresponding emulsion reservoir whencarrier fluid is supplied to the carrier port and vacuum is applied tothe vacuum port; wherein the device includes a plurality of layersbonded together and forming the sample reservoirs, the sample ports, theemulsion reservoirs, the carrier port, the vacuum port, and the channelnetwork, and wherein each of the sample, carrier, and vacuum portsincludes a top rim formed by a protrusion.

89. The device of paragraph 88, wherein the device includes one or moreprotrusions that collectively encircles a central axis of each port.

90. The device of paragraph 88 or paragraph 89, wherein all of the rimsare coplanar with one another.

91. The device of any of paragraphs 88 to 90, further comprising arespective vent port for each sample reservoir, the vent portcommunicating with the sample reservoir via the corresponding sampleport and including a rim that is coplanar with some or all of the otherrims.

92. The device of any of paragraphs 88 to 91, wherein the plurality oflayers includes a layer having a body, and wherein each rim is formed bya protrusion that protrudes from the body.

93. The device of paragraph 92, wherein the body has a substantiallyplanar surface and wherein each protrusion protrudes from the planarsurface.

94. The device of any of paragraphs 88 to 93, further comprising asealing layer configured to be bonded to each rim, such that the sealinglayer prevents any fluid communication between the ambient atmosphereoutside the device and each sample reservoir, each emulsion reservoir,and the channel network.

95. A device to form an array of emulsions, comprising: (A) an array ofsample reservoirs; (B) an array of sample ports overlying and alignedwith the array of sample reservoirs; (C) an array of emulsion reservoirscorresponding to the array of sample reservoirs; (D) a carrier port; (E)a vacuum port; (F) an array of vent ports, each vent port being in fluidcommunication with one of the sample reservoirs via a correspondingsample port; and (G) a channel network in fluid communication with eachof the sample and emulsion reservoirs and with the carrier port andconfigured to form droplets of sample fluid from each sample reservoirand direct the droplets to a corresponding emulsion reservoir whencarrier fluid is supplied to the carrier port and vacuum is applied tothe vacuum port; wherein the device includes a plurality of layersbonded together and forming the sample reservoirs, the sample ports, theemulsion reservoirs, the carrier port, the vacuum port, the vent ports,and the channel network.

96. The device of paragraph 95, wherein one of the layers is a moldedlayer defining a plurality of through-holes, and wherein thethrough-holes form at least a portion of each sample port and at least aportion of each vent port.

97. A method of forming an array of emulsions, the method comprising:(A) selecting a microfluidic device composed of a plurality of layersbonded together, the layers forming a plurality of sample reservoirs, acorresponding plurality of emulsion reservoirs, and a channel networkproviding fluid communication between the sample reservoirs and theemulsion reservoirs; (B) dispensing sample fluid into each samplereservoir; (C) bonding a sealing layer to a plurality of coplanarprotrusions located on a top side of the device, wherein the step ofbonding fluidically isolates each sample reservoir from the ambientatmosphere outside the device; (D) forming one or more openings throughthe sealing layer; (E) supplying carrier fluid to the channel network;and (F) applying vacuum or positive pneumatic pressure at each of theone or more openings to drive sample fluid from each sample reservoir toa channel intersection of the channel network, at which droplets of thesample fluid enclosed by carrier fluid are formed, and from which thedroplets are directed to a corresponding emulsion reservoir.

Example 5. Selected Embodiments II

This example describes further selected embodiments of the presentdisclosure presented as a series of indexed paragraphs.

Paragraph A1 A device for forming emulsions, comprising: (i) a frame;and (ii) a plurality of separate microfluidic modules mounted to theframe, each module including an array of emulsion formation units, eachemulsion formation unit including a sample reservoir configured to beloaded with sample-containing fluid, and a droplet generation siteconfigured to receive sample-containing fluid from the sample reservoirand to generate droplets of sample-containing fluid encapsulated bycarrier fluid; and (iii) a same sealing member configured to be bondedto a top side of each module of the plurality of modules, such that thesealing member covers and seals a plurality of ports of each module,each port being in fluid communication with the emulsion formation unitsof the module.

Paragraph A2. The device of paragraph A1, wherein the plurality of portsof each module is defined by a port layer including a body and at leastone protrusion projecting from a top surface of the body, wherein thebody defines a lower section of each port of the plurality of ports,wherein the at least one protrusion forms a top rim of each port of theplurality of ports, and wherein the sealing member is bonded directly tothe top rim of each port.

Paragraph A3. The device of paragraph A2, wherein the top rim of eachport is elevated with respect to a top of the frame.

Paragraph A4. The device of any one of paragraphs A1 to A3, wherein eachmodule is attached to the frame via complementary mating features of themodule and the frame.

Paragraph A5. The device of paragraph A4, wherein each module is mountedto the frame via snap-fit attachment.

Paragraph A6. The device of any one of paragraphs A1 to A5, wherein thesealing member is configured to hermetically seal each module, such thateach of the emulsion formation units of the module is isolated from theambient atmosphere outside the module.

Paragraph A7. The device of any one of paragraphs A1 to A6, wherein eachsample reservoir is located under a respective sample-loading port, andwherein the sample-loading port includes a tapered region that taperstoward the sample reservoir.

Paragraph A8. The device of paragraph A7, wherein a neck region of thesample-loading port is located between the tapered region and the samplereservoir.

Paragraph A9. The device of any one of paragraphs A1 to A8, wherein eachmodule includes a vacuum port and a vacuum manifold, and wherein thevacuum port is in fluid communication with an emulsion reservoir of eachemulsion formation unit of the module via the vacuum manifold.

Paragraph A10. The device of any one of paragraphs A1 to A9, wherein thearray of each module is a smaller array, and wherein the plurality ofmodules forms a larger, rectangular array of emulsion formation unitsthat includes the smaller array of each module.

Paragraph A11. The device of paragraph A10, wherein each samplereservoir is configured to be loaded with sample-containing fluid via arespective sample-loading port that defines a loading axis, and whereinthe rectangular array has the loading axes of the sample-loading portsarranged in uniformly-spaced rows and uniformly-spaced columns.

Paragraph A12. The device of paragraph A11, wherein the rows and thecolumns have the same spacing as one another.

Paragraph A13. The device of any one of paragraphs A10 to A12, whereinthe rectangular array is an 8-by-12 array.

Paragraph A14. The device of any one of paragraphs A1 to A13, whereineach module includes at least one carrier fluid port and at least onevacuum/pressure port, and wherein the emulsion formation units of themodule are configured to form and collect an array of emulsions whencarrier fluid is supplied to the at least one carrier fluid port andvacuum or pressure is applied to each port of the at least onevacuum/pressure port.

Paragraph A15. The device of paragraph A14, wherein the at least onevacuum/pressure port is a single vacuum port, and wherein the moduleincludes a vacuum manifold configured to communicate vacuum applied atthe vacuum port to each emulsion formation unit of the module.

Paragraph A16. The device of paragraph A15, wherein the at least onecarrier fluid port is a single carrier fluid port, and wherein themodule includes a carrier fluid manifold that directs carrier fluid fromthe single carrier fluid port to the droplet-generation site of eachemulsion formation unit of the module.

Paragraph A17. The device of any one of paragraphs A1 to A16, whereineach emulsion formation unit of the plurality of modules includes anemulsion reservoir to collect an emulsion including droplets ofsample-containing fluid encapsulated by carrier fluid, and wherein thedroplet generation site of the emulsion formation unit includes achannel junction at which a sample channel, at least one carrier fluidchannel, and a droplet channel meet one another and at which dropletsare generated.

Paragraph A18. The device of any one of paragraphs A1 to A17, whereinthe sealing member includes a heat-sealable foil.

Paragraph A19. The device of any one of paragraphs A1 to A18, whereinthe plurality of ports includes a plurality of sample-loading ports, atleast one carrier fluid port, and at least one vacuum/pressure port.

Paragraph A20. The device of paragraph A19, wherein the plurality ofports also includes a respective vent port for each emulsion formationunit.

Paragraph A21. The device of any one of paragraphs A1 to A20, whereineach module defines a carrier fluid port that is in fluid communicationwith the droplet generation site of each emulsion formation unit of themodule, and wherein the carrier fluid ports of the plurality of modulesare arranged along a straight line.

Paragraph A22. The device of any one of paragraphs A1 to A21, whereineach module includes an assembly of layers bonded together, and whereinthe assembly creates each emulsion formation unit of the module.

Paragraph A23. A system for forming emulsions, comprising: (i) thedevice of any one of paragraphs A1 to A22; and (ii) at least one sourceof vacuum/pressure configured to be operatively coupled to the device,to drive droplet generation at each droplet generation site of eachmodule.

Paragraph A24. The system of paragraph A23, wherein the at least onesource of vacuum/pressure is a vacuum source configured to beoperatively coupled to each module.

Paragraph A25. The system of paragraph A23 or paragraph 24, furthercomprising at least one source of carrier fluid configured to beoperatively coupled to the device to supply carrier fluid to eachmodule.

Paragraph A26. The system of paragraph A25, wherein the at least onesource of vacuum/pressure and the at least source of carrier fluid areprovided by the same instrument.

Paragraph A27. A method of forming emulsions, the method comprising: (i)selecting a device including a frame and a plurality of separatemicrofluidic modules mounted to the frame, each module including anarray of emulsion formation units; (ii) loading emulsion formation unitsof each array with sample-containing fluid; (iii) bonding a same sealingmember to each module of the plurality of modules; and (iv) applyingvacuum and/or pressure through the sealing member to the array of eachmodule, to generate droplets of sample-containing fluid encapsulated bycarrier fluid, using emulsion formation units of the module.

Paragraph A28. The method of paragraph A27, wherein the step of applyingvacuum and/or pressure includes a step of applying vacuum to a singleline of vacuum ports defined collectively by the plurality of modules.

Paragraph A29. The method of paragraph A27 or paragraph A28, wherein thestep of applying vacuum and/or pressure is performed through openingsdefined by the sealing member, and wherein the openings are createdafter the step of bonding.

Paragraph A30. The method of any one of paragraphs A27 to A29, furthercomprising a step of supplying carrier fluid through the sealing memberto the array of each module.

Paragraph A31. The method of paragraph A30, wherein the step ofsupplying carrier fluid includes a step of supplying carrier fluid to asingle line of carrier fluid ports defined collectively by the pluralityof modules.

Paragraph A32. The method of any one of paragraphs A27 to A31, whereinthe step of bonding includes a step of bonding the sealing memberdirectly to one or more protrusions formed on a top side of each module,and wherein the one or more protrusions form a top rim of a plurality ofports of the module.

Paragraph A33. The method of any one of paragraph A27 to A32, furthercomprising any limitation or combination of limitations of paragraphs A1to A26.

Paragraph A34. A method of forming an array of emulsions, the methodcomprising: (i) selecting the device or system of any one of paragraphsA1 to A26; (ii) loading sample-containing fluid into sample reservoirsof each module, optionally via corresponding, overlying sample-loadingports, and optionally using a respective pipette tip placed into eachsample reservoir via a corresponding sample-loading port; (iii)optionally, sealing ports on a top side of each module, optionally withthe same sealing member, and, optionally with the sealing member bondeddirectly to a projecting top rim of each port; (iv) optionally, formingopenings in the sealing member, optionally by piercing, to access,through the sealing member, at least one carrier fluid port of eachmodule, at least one vacuum/pressure port of each module, a respectivevent port for each emulsion formation unit, or any combination thereof;(v) supplying carrier fluid to droplet generation sites of each module,optionally via the at least one carrier fluid port of the module,optionally from at least one external (or internal) carrier fluidsource, optionally to all of the modules in parallel, and optionally toonly a single line of carrier fluid ports of the modules; (vi) applyingvacuum or pressure to each vacuum/pressure port of each module,optionally to all of the modules in parallel, and optionally to only asingle line of vacuum/pressure ports of the modules; (vii) generatingsample-containing droplets encapsulated with carrier fluid at a dropletgeneration site of each module; and (viii) optionally, collecting anemulsion including sample-containing droplets in an emulsion reservoirof each emulsion formation unit.

Paragraph A35. The device, system, or method of any one of paragraphs A1to A34, further comprising any limitation or combination of limitationsof Example 4.

Paragraph B1. A device to form an array of emulsions, comprising: anassembly of bonded layers creating an array of emulsion formation units,each emulsion formation unit including a sample reservoir configured tobe loaded with sample-containing fluid, and a droplet generation siteconfigured to receive sample-containing fluid from the sample reservoirand to generate droplets of sample-containing fluid encapsulated bycarrier fluid, the layers including a port layer forming a plurality ofports arranged in fluid communication with the array, wherein the portlayer includes a body and at least one protrusion projecting upwardlyfrom the body, and wherein the at least one protrusion forms anencircling rim of each port.

Paragraph B2. The device of paragraph B1, wherein the body defines alower section of each port.

Paragraph B3. The device of paragraph B1 or paragraph B2, wherein eachemulsion formation unit includes an emulsion reservoir to collect anemulsion including droplets of sample-containing fluid encapsulated bycarrier fluid, and wherein the droplet generation site includes achannel junction at which a sample channel, at least one carrier fluidchannel, and a droplet channel meet one another and at which dropletsare generated.

Paragraph B4. The device of any one of paragraphs B1 to B3, wherein theat least one protrusion projects from a top surface of the body.

Paragraph B5. The device of paragraph B4, wherein the top surface liesin a plane.

Paragraph B6. The device of any one of paragraphs B1 to B5, wherein theassembly defines a plane, wherein the body and the at least oneprotrusion each have a thickness measured orthogonal to the plane, andwherein the thickness of the body is greater than the thickness of theat least one protrusion.

Paragraph B7. The device of any one of paragraphs B1 to B6, wherein theencircling rims of the plurality of ports are coplanar with one anotheron top.

Paragraph B8. The device of any one of paragraphs B1 to B7, furthercomprising a sealing member configured to be bonded directly to eachencircling rim such that each port of the plurality of ports is coveredand sealed.

Paragraph B9. The device of any one of paragraphs B1 to B8, wherein theplurality of ports includes a respective sample-loading port overlyingeach sample reservoir, and wherein the sample-loading port includes atapered region that tapers toward the sample reservoir.

Paragraph B10. The device of paragraph B9, wherein a neck region of thesample-loading port is located between the tapered region and the samplereservoir.

Paragraph B11. The device of any one of paragraphs B1 to B10, whereinthe port layer is a molded layer defining a plurality of through-holes,and wherein one of the through-holes forms at least part of each port.

Paragraph B12. The device of any one of paragraphs B1 to B11, furthercomprising any limitation or combination of limitations of paragraphs A1to A34 and Example 4.

Paragraph C1. A device to form an array of emulsions, comprising: anassembly of bonded layers creating an array of emulsion formation units,each emulsion formation unit including a sample reservoir configured tobe loaded with sample-containing fluid, and a droplet generation siteconfigured to receive sample-containing fluid from the sample reservoirand to generate droplets of sample-containing fluid encapsulated bycarrier fluid; wherein the assembly includes a vacuum port and a vacuummanifold, and wherein the vacuum port is in fluid communication with thedroplet generation site of each emulsion formation unit via the vacuummanifold.

Paragraph C2. The device of paragraph C1, wherein each emulsionformation unit includes an emulsion reservoir to collect an emulsionincluding droplets of sample-containing fluid encapsulated by carrierfluid, and wherein the vacuum port is in fluid communication with theemulsion reservoir via the vacuum manifold.

Paragraph C3. The device of paragraph C1 or C2, wherein the dropletgeneration site includes a channel junction at which a sample channel,at least one carrier fluid channel, and a droplet channel meet oneanother and at which droplets are generated,

Paragraph C4. The device of any one of paragraphs C1 to C3, wherein theassembly includes a carrier fluid port and a carrier manifold, andwherein the carrier fluid port is in fluid communication with eachdroplet generation site via the carrier manifold.

Paragraph C5. A device for forming emulsions, comprising: (i) an arrayof sample reservoirs; (ii) an array of sample-loading ports overlyingand aligned with the array of sample reservoirs; (iii) an array ofemulsion reservoirs corresponding to the array of sample reservoirs;(iv) a carrier fluid port; (v) at least one vacuum/pressure port; (vi) avacuum/pressure manifold in fluid communication with each emulsionreservoir; and (vii) a channel network in fluid communication with eachof the sample and emulsion reservoirs and with the carrier port andconfigured to form droplets of sample-containing fluid received fromeach sample reservoir and to direct the droplets to a correspondingemulsion reservoir when carrier fluid is supplied to the carrier fluidport and vacuum or positive pneumatic pressure is applied to each portof the at least one vacuum/pressure port; wherein the device includes aplurality of layers bonded together and forming the sample reservoirs,the sample ports, the emulsion reservoirs, the carrier fluid port, thevacuum/pressure port, the vacuum/pressure manifold, and the channelnetwork.

Paragraph C6. The device of any one of paragraphs C1 to C5, furthercomprising any limitation or combination of limitations of paragraphs A1to A34, B1 to B11, and Example 4.

Paragraph D1. A device for forming emulsions, comprising: an assembly ofbonded layers creating an array of emulsion formation units, eachemulsion formation unit including a sample reservoir configured to beloaded with sample-containing fluid, and a droplet generation siteconfigured to receive sample-containing fluid from the sample reservoirand to generate droplets of sample-containing fluid encapsulated bycarrier fluid; wherein a lower portion of the assembly forms the samplereservoir and the droplet generation site of each emulsion formationunit, wherein an upper portion of the assembly defines a respectivesample-loading port overlying each sample reservoir, and wherein eachsample-loading port includes a tapered region that tapers toward thesample reservoir.

Paragraph D2. The device of paragraph D1, wherein the sample-loadingport forms a neck region under the tapered region, and wherein the neckregion has a smaller average diameter than the tapered region or thesample reservoir.

Paragraph D3. The device of paragraph D1 or paragraph D2, wherein theupper portion of the assembly includes a port layer having a body and atleast one protrusion, wherein the at least one protrusion projectsupwardly from the body and forms a respective encircling rim of eachsample-loading port.

Paragraph D4. The device of any one of paragraphs D1 to D3, wherein theassembly defines a plane, and wherein the sample-loading port taperssuch that a maximum cross-sectional area of the sample loading porttaken parallel to the plane is at least three times a minimumcross-sectional area of the sample-loading port taken parallel to theplane.

Paragraph D5. The device of any one of paragraphs D1 to D4, wherein thesample-loading port has a maximum diameter and a minimum diameter, andwherein the maximum diameter is at least about twice the minimumdiameter.

Paragraph D6. The device of any one of paragraphs D1 to D5, wherein theassembly defines a plane, and wherein a cross-sectional shape of thesample-loading port parallel to the plane changes between the top andthe bottom of the tapered region.

Paragraph D7. The device of any one of paragraphs D1 to D6, wherein theassembly defines a plane, and wherein at least a portion of the taperedregion forms an angle of less than about 60 degrees with the plane.

Paragraph D8. The device of any one of paragraphs D1 to D7, furthercomprising any limitation or combination of limitations of paragraphs A1to A34, B1 to B11, C1 to C5, and Example 4.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions of the present disclosure.Further, ordinal indicators, such as first, second, or third, foridentified elements are used to distinguish between the elements, and donot indicate a particular position or order of such elements, unlessotherwise specifically stated.

We claim:
 1. A method of forming emulsions, the method comprising:selecting a device including a frame and a plurality of discretemicrofluidic modules, each microfluidic module being mounted to theframe and including an array of emulsion formation units; loadingemulsion formation units of each array with sample-containing fluid;selecting a sealing member; bonding the sealing member to eachmicrofluidic module of the plurality of microfluidic modules; andapplying vacuum and/or pressure through the sealing member to the arrayof emulsion formation units of each microfluidic module, to generatedroplets of sample-containing fluid encapsulated by carrier fluid, usingemulsion formation units of the microfluidic module.
 2. The method ofclaim 1, wherein the device selected has each microfluidic moduleattached to the frame via complementary mating features of themicrofluidic module and the frame.
 3. The method of claim 2, furthercomprising mounting each microfluidic module to the frame via snap-fitattachment before selecting.
 4. The method of claim 1, wherein eachemulsion formation unit includes a sample reservoir and a sample-loadingport overlying the sample reservoir, wherein loading includes loadingsample-containing fluid into the sample reservoir via the sample-loadingport, and sealing includes sealing an opening of the sample-loading portto be fluid-tight using the sealing member, such that fluid cannot enteror exit the sample-loading port via the opening.
 5. The method of claim1, wherein bonding includes bonding the sealing member directly to oneor more protrusions formed on a top side of each microfluidic module,and wherein the one or more protrusions form a top rim of a plurality ofports of the microfluidic module.
 6. The method of claim 1, wherein eachemulsion formation unit has a plurality of ports each defining anopening, and wherein bonding includes sealing the opening of each portof the plurality of ports to be fluid-tight using the sealing member,such that fluid cannot enter or exit the port via the opening.
 7. Themethod of claim 6, wherein the plurality of ports includes a pluralityof sample-loading ports, at least one carrier fluid port, and at leastone vacuum/pressure port.
 8. The method of claim 7, wherein theplurality of ports also includes a respective vent port for eachemulsion formation unit.
 9. The method of claim 1, further comprisingforming openings in the sealing member after bonding, to access, throughthe sealing member, at least one carrier fluid port of each microfluidicmodule, at least one vacuum/pressure port of the microfluidic module,and a respective vent port for each emulsion formation unit of themicrofluidic module.
 10. The method of claim 9, wherein forming openingsincludes piercing the sealing member.
 11. The method of claim 1, whereinapplying vacuum and/or pressure includes applying vacuum to only asingle line of vacuum ports defined collectively by the plurality ofmicrofluidic modules.
 12. The method of claim 1, wherein eachmicrofluidic module has a plurality of sample reservoirs to receivesample-containing fluid and forms a channel network in which droplets ofsample-containing fluid are formed, and wherein the channel network islocated above a lower portion of the sample reservoir of themicrofluidic module.
 13. The method of claim 1, wherein eachmicrofluidic module includes a plurality of layers nonremovably bondedto one another and forming each emulsion formation unit of themicrofluidic module, and wherein applying pressure/vacuum includesapplying pressure or vacuum to each emulsion formation unit of themicrofluidic module via a manifold created by one or more layers of theplurality of layers.
 14. The method of claim 13, wherein eachmicrofluidic module includes a plurality of sample reservoirs to receivesample-containing fluid, wherein the plurality of layers includes a welllayer forming a lower portion of each sample reservoir of the pluralityof sample reservoirs, a channel layer located over the well layer andforming a droplet generation site of each emulsion formation unit of themicrofluidic module, and a port layer located over the channel layer andforming a plurality of ports arranged in fluid communication with theemulsion formation units of the microfluidic module, and whereinapplying pressure/vacuum includes applying pressure or vacuum to eachemulsion formation unit of the microfluidic module via a vacuum/pressureport formed by the port layer.
 15. The method of claim 14, wherein theport layer defines a respective sample-loading port over the samplereservoir of each emulsion formation unit of the microfluidic module, avent port for the sample reservoir, and a channel that fluidicallyconnects the vent port to the sample-loading port, wherein the portlayer includes a body having a top surface and also includes at leastone protrusion projecting upwardly from the top surface and formedintegrally with the body, wherein the at least one protrusion forms anencircling rim of each sample-loading port and each vent port, andwherein sealing includes bonding the sealing member directly to theencircling rim of each sample-loading port and each vent port.